Coronavirus vaccine

ABSTRACT

This disclosure relates to the field of RNA to prevent or treat coronavirus infection. In particular, the present disclosure relates to methods and agents for vaccination against coronavirus infection and inducing effective coronavirus antigen-specific immune responses such as antibody and/or T cell responses. Specifically, in one embodiment, the present disclosure relates to methods comprising administering to a subject RNA encoding a peptide or protein comprising an epitope of SARS-CoV-2 spike protein (S protein) for inducing an immune response against coronavirus S protein, in particular S protein of SARS-CoV-2, in the subject, i.e., vaccine RNA encoding vaccine antigen.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in .xml format and is hereby incorporated byreference in its entirety. Said .xml file, created on Apr. 11, 2023, isidentified as 2013237-0518 and is 453,809 bytes in size.

This disclosure relates to the field of RNA to prevent or treatcoronavirus infection. In particular, the present disclosure relates tomethods and agents for vaccination against coronavirus infection andinducing effective coronavirus antigen-specific immune responses such asantibody and/or T cell responses. These methods and agents are, inparticular, useful for the prevention or treatment of coronavirusinfection. Administration of RNA disclosed herein to a subject canprotect the subject against coronavirus infection. Specifically, in oneembodiment, the present disclosure relates to methods comprisingadministering to a subject RNA encoding a peptide or protein comprisingan epitope of SARS-CoV-2 spike protein (S protein) for inducing animmune response against coronavirus S protein, in particular S proteinof SARS-CoV-2, in the subject, i.e., vaccine RNA encoding vaccineantigen. Administering to the subject RNA encoding vaccine antigen mayprovide (following expression of the RNA by appropriate target cells)vaccine antigen for inducing an immune response against vaccine antigen(and disease-associated antigen) in the subject.

Coronaviruses are positive-sense, single-stranded RNA ((+)ssRNA)enveloped viruses that encode for a total of four structural proteins,spike protein (S), envelope protein (E), membrane protein (M) andnucleocapsid protein (N). The spike protein (S protein) is responsiblefor receptor-recognition, attachment to the cell, infection via theendosomal pathway, and the genomic release driven by fusion of viral andendosomal membranes. Though sequences between the different familymembers vary, there are conserved regions and motifs within the Sprotein making it possible to divide the S protein into two subdomains:S1 and S2. While the S2, with its transmembrane domain, is responsiblefor membrane fusion, the S1 domain recognizes the virus-specificreceptor and binds to the target host cell. Within several coronavirusisolates, the receptor binding domain (RBD) was identified and a generalstructure of the S protein defined (FIG. 1 ).

In December 2019, a pneumonia outbreak of unknown cause occurred inWuhan, China and it became clear that a novel coronavirus (severe acuterespiratory syndrome coronavirus 2; SARS-CoV-2) was the underlyingcause. The genetic sequence of SARS-CoV-2 became available to the WHOand public (MN908947.3) and the virus was categorized into thebetacoronavirus subfamily. By sequence analysis, the phylogenetic treerevealed a closer relationship to severe acute respiratory syndrome(SARS) virus isolates than to another coronavirus infecting humans,namely the Middle East respiratory syndrome (MERS) virus.

SARS-CoV-2 infections and the resulting disease COVID-19 have spreadglobally, affecting a growing number of countries. On 11 Mar. 2020 theWHO characterized the COVID-19 outbreak as a pandemic. As of 1 Dec.2020, there have been >63 million globally confirmed COVID-19 casesand >1.4 million deaths, with 191 countries/regions affected. Theongoing pandemic remains a significant challenge to public health andeconomic stability worldwide.

Every individual is at risk of infection as there is no pre-existingimmunity to SARS-CoV-2. Following infection some but not all individualsdevelop protective immunity in terms of neutralising antibody responsesand cell mediated immunity. However, it is currently unknown to whatextent and for how long this protection lasts. According to WHO 80% ofinfected individuals recover without need for hospital care, while 15%develop more severe disease and 5% need intensive care. Increasing ageand underlying medical conditions are considered risk factors fordeveloping severe disease.

The presentation of COVID-19 is generally with cough and fever, withchest radiography showing ground-glass opacities or patchy shadowing.However, many patients present without fever or radiographic changes,and infections may be asymptomatic which is relevant to controllingtransmission. For symptomatic subjects, progression of disease may leadto acute respiratory distress syndrome requiring ventilation andsubsequent multi-organ failure and death. Common symptoms inhospitalized patients (in order of highest to lowest frequency) includefever, dry cough, shortness of breath, fatigue, myalgias,nausea/vomiting or diarrhoea, headache, weakness, and rhinorrhoea.Anosmia (loss of smell) or ageusia (loss of taste) may be the solepresenting symptom in approximately 3% of individuals who have COVID-19.

All ages may present with the disease, but notably case fatality rates(CFR) are elevated in persons >60 years of age. Comorbidities are alsoassociated with increased CFR, including cardiovascular disease,diabetes, hypertension, and chronic respiratory disease. Healthcareworkers are overrepresented among COVID-19 patients due to occupationalexposure to infected patients.

In most situations, a molecular test is used to detect SARS-CoV-2 andconfirm infection. The reverse transcription polymerase chain reaction(RT-PCR) test methods targeting SARS-CoV-2 viral RNA are the goldstandard in vitro methods for diagnosing suspected cases of COVID-19.Samples to be tested are collected from the nose and/or throat with aswab.

Among other things, the present disclosure provides insights into immuneresponses elicited by exposure to (e.g., by vaccination and/orinfection) different SARS-CoV-2 variants or immunogenic polypeptides(e.g., S protein), or immunogenic fragments thereof. For example, insome embodiments, administering RNA encoding an S protein of a BA.2and/or BA.4/5 Omicron SARS-CoV-2 variant, or an immunogenic fragmentthereof, can result in an improved immune response, which includes,e.g., improved neutralization of Omicron BA.4 and/or Omicron BA.5SARS-CoV-2 variants and/or broader cross-neutralization of Omicronvariants of concern (e.g., higher neutralization titers against a largernumber of Omicron variants of concern). In some embodiments, the presentdisclosure provides an insight that a bivalent coronavirus vaccine(e.g., a bivalent BA.4/5 vaccine comprising a first RNA encoding aSARS-CoV-2 S protein of a Wuhan strain or an immunogenic fragmentthereof, and a second RNA encoding a SARS-CoV-2 S protein comprising oneor more mutations that are characteristic of a BA.4/5 Omicron variant oran immunogenic fragment thereof) can provide broadercross-neutralization against SARS-CoV-2 Wuhan strain and certainvariants thereof (e.g., in some embodiments variants that are prevalentand/or rapidly spreading in a relevant jurisdiction, e.g., certainOmicron variants) in certain subjects as compared to a monovalentcoronavirus vaccine (e.g., a vaccine comprising RNA encoding aSARS-CoV-2 S protein of a coronavirus strain or variant thereof). Insome embodiments, such broader cross-neutralization can be observed invaccine-naïve subjects. In some embodiments, such broadercross-neutralization can be observed in subjects without a coronavirusinfection (e.g., a SARS-CoV-2 infection). In some embodiments, suchbroader cross-neutralization can be observed in subjects who previouslyreceived a SARS-CoV-2 vaccine (e.g., in some embodiments an RNA vaccineencoding a SARS-CoV-2 S protein, e.g., in some embodiments of a Wuhanstrain). In some embodiments, such broader cross-neutralization can beobserved in in young pediatric subjects (e.g., subjects aged 6 months toless than 2 years, and/or 2 years to less than 5 years). In someembodiments, the present disclosure provides an insight that exposure toat least two certain SARS-CoV-2 variants or immunogenic polypeptides(e.g., S protein), or immunogenic fragments thereof can result in asynergistic improvement in immune response (e.g., higher neutralizationtiters, broader cross-neutralization, and/or an immune response that isless susceptible to immune escape) as compared to exposure to oneSARS-CoV-2 strain and/or other combination of SARS-CoV-2 variants. Insome embodiments, the present disclosure provides an insight thatexposure to a SARS-CoV-2 S protein from a Wuhan strain or an immunogenicfragment thereof (e.g., by vaccination and/or infection), and exposureto a SARS-CoV-2 S protein of an Omicron BA.1 variant or an immunogenicfragment thereof (e.g., by vaccination and/or infection) can result in asynergistic improvement in immune response (e.g., higher neutralizationtiters, broader cross-neutralization, and/or an immune response that isless susceptible to immune escape) as compared to exposure to oneSARS-CoV-2 strain and/or other combinations of SARS-CoV-2 variants. Insome embodiments, the present disclosure provides an insight thatexposure to a SARS-CoV-2 S protein from a Wuhan strain or an immunogenicfragment thereof (e.g., by vaccination and/or infection), and exposureto a SARS-CoV-2 S protein of an Omicron BA.4 or BA.5 variant or animmunogenic fragment thereof (e.g., by vaccination and/or infection) canresult in an synergistic improvement in immune response (e.g., higherneutralization titers, broader cross-neutralization, and/or an immuneresponse that is less susceptible to immune escape) as compared toexposure to one SARS-CoV-2 strain and/or other combinations ofSARS-CoV-2 variants. In some embodiments, the present disclosureprovides an insight that (i) exposure to a SARS-CoV-2 S protein from astrain/variant selected from the group consisting of Wuhan strain, analpha variant, beta variant, delta variant, Omicron BA.1, andsublineages derived from any of the aforementioned strains/variants, orimmunogenic fragments thereof (e.g., by vaccination and/or infection),combined with (ii) exposure to a SARS-CoV-2 S protein from astrain/variant selected from the group consisting of Omicron BA.2,Omicron BA.4, Omicron BA.5, and sublineages derived from any of theaforementioned strains/variants, or immunogenic fragments thereof (e.g.,by vaccination and/or infection) can result in a synergistic improvementin immune response (e.g., higher neutralization titers, broadercross-neutralization, and/or an immune response that is less susceptibleto immune escape) as compared to exposure to one SARS-CoV-2 strainand/or other combinations of SARS-CoV-2 variants).

The present disclosure also provides significant insights into how animmune response develops in subjects following exposures to (e.g.,vaccinations and/or infections) multiple, different SARS-CoV-2 strains.Among other things, disclosed herein is a finding that differentcombinations of SARS-CoV-2 variants elicit different immune responses.Specifically, the present disclosure provides an insight that exposureto certain combinations of SARS-CoV-2 variants can elicit an improvedimmune response (e.g., higher neutralization titers, broadercross-neutralization, and/or an immune response that is less susceptibleto immune escape). In some embodiments, an improved immune response canbe produced when subjects are delivered two or more antigens (e.g., aspolypeptides or RNAs encoding such polypeptides), each having few sharedepitopes. In some embodiments, an improved immune response can beproduced when subjects are delivered a combination of SARS-CoV-2 Sproteins (e.g., as polypeptides or RNAs encoding such polypeptides)sharing no more than 50% (e.g., no more than 40%, no more than 30%, nomore 20% or more) of epitopes (including, e.g., amino acid mutations)that can be bound by neutralization antibodies. In some embodiments, animproved immune response can be produced by delivering, as polypeptidesor RNAs encoding such polypeptides, (a) a SARS-CoV-2 S protein from aWuhan strain, an Alpha variant, Beta variant, or a Delta variant ofSARS-CoV-2 or an immunogenic fragment thereof, and (b) an S protein froma SARS-CoV-2 Omicron variant or an immunogenic fragment thereof. In someembodiments, an improved immune response can be produced by delivering,as polypeptides or RNAs encoding such polypeptides, (a) a SARS-CoV-2 Sprotein from a Wuhan strain, an Alpha variant, a Beta variant, or aDelta variant of SARS-CoV-2 or an immunogenic fragment thereof, and (b)an S protein of a SARS-CoV-2 Omicron variant that is not a BA.1 Omicronvariant or an immunogenic fragment thereof. In some embodiments, animproved immune response can be produced by delivering, as polypeptidesor RNAs encoding such polypeptides, (a) an S protein from a Wuhanstrain, an Alpha variant, a Beta Variant, a Delta SARS-CoV-2 variant, ora BA.1 Omicron variant or an immunogenic fragment thereof and (b) an Sprotein of a SARS-CoV-2 Omicron variant that is not a BA.1 Omicronvariant or an immunogenic fragment thereof. In some embodiments, animproved immune response can be produced by delivering, as polypeptidesor RNAs encoding such polypeptides, (a) a SARS-CoV-2 S protein from aWuhan strain, an Alpha variant, a Beta variant, or a Delta variant, oran immunogenic fragment thereof and (b) an S protein of a BA.2 or a BA.4or BA.5 SARS-CoV-2 Omicron variant or an immunogenic fragment thereof.

In some embodiments, the present disclosure also provides an insightthat administration of multiple doses (e.g., at least 2, at least 3, atleast 4, or more doses) of a coronavirus vaccine described herein (e.g.,a bivalent vaccine described herein such as a bivalent BA.4/5 vaccine)may provide certain beneficial effect(s) on affinity of antibodiesagainst one or more SARS-CoV-2 strain or variants thereof. In someembodiments, such beneficial effect(s) on affinity of antibodies may beobserved with respect to antibodies against certain Omicron variants. Byway of example only, in some embodiments, such beneficial effect(s) onaffinity of antibodies may be observed with respect to antibodiesagainst certain Omicron variants that share at least one or more commonepitopes, for example, with a Wuhan strain.

Also disclosed herein are compositions that can produce an improvedimmune response (e.g., an immune response having broadercross-neutralization activity, stronger neutralization, and/or which isless susceptible to immune escape). In some embodiments, a compositiondescribed herein comprises two or more antigens or nucleic acids (e.g.,RNA) that encodes such antigens that have few shared epitopes. In someembodiments, a composition described herein delivers, as polypeptides ornucleic acids encoding such polypeptides, a combination of SARS-CoV-2 Sproteins or immunogenic fragments thereof sharing no more than 50%(e.g., no more than 40%, no more than 30%, no more than 20% or more) ofepitopes (including, e.g., amino acid mutations) that can be bound byneutralization antibodies. In some embodiments, a composition describedherein comprises (a) RNA encoding a SARS-CoV-2 S protein from a Wuhanstrain, an Alpha variant, a Beta variant, or a Delta variant or animmunogenic fragment thereof and (b) RNA encoding an S protein from anOmicron variant of SARS-CoV-2 (e.g., in some embodiments an S proteinfrom a BA.1, BA.2, or BA.4/5 Omicron variant) or an immunogenic fragmentthereof. In some embodiments, a composition described herein comprises(a) RNA encoding a SARS-CoV-2 S protein from a Wuhan strain, an Alphavariant, a Beta variant, or a Delta variant or an immunogenic fragmentthereof and (b) RNA encoding an S protein of an Omicron variant ofSARS-CoV-2 that is not a BA.1 Omicron variant or an immunogenic fragmentthereof. In some embodiments, a composition described herein comprises(a) RNA encoding a SARS-CoV-2 S protein of a Wuhan strain, an Alphavariant, a Beta variant, or a Delta variant or a BA.1 Omicron variant oran immunogenic fragment thereof and (b) RNA encoding an S protein of aOmicron variant that is not a BA.1 Omicron variant or an immunogenicfragment thereof. In some embodiments, a composition described hereincomprises (a) RNA encoding a SARS-CoV-2 S protein of a Wuhan strain, anAlpha variant, a Beta variant or a Delta variant of SARS-CoV-2 and (b)RNA encoding an S protein from a BA.2 or a BA.4 or BA.5 Omicron variantof SARS-CoV-2 or an immunogenic fragment thereof. In some embodiments, acomposition described herein comprises RNA encoding an S protein from aBA.2 Omicron variant of SARS-CoV-2 or an immunogenic fragment thereof.In some embodiments, a composition comprises RNA encoding an S proteinfrom a BA.4 or BA.5 Omicron variant of SARS-CoV-2 or an immunogenicfragment thereof. SARS-CoV-2 is an RNA virus with four structuralproteins. One of them, the spike protein is a surface protein whichbinds the angiotensin-converting enzyme 2 (ACE-2) present on host cells.Therefore, the spike protein is considered a relevant antigen forvaccine development. BNT162b2 (SEQ ID NO: 20) is an mRNA vaccine forprevention of COVID-19 and demonstrated an efficacy of 95% or more atpreventing COVID-19. The vaccine is made of a 5′capped mRNA encoding forthe full-length SARS-CoV-2 spike glycoprotein (S) encapsulated in lipidnanoparticles (LNPs). The finished product is presented as a concentratefor dispersion for injection containing BNT162b2 as active substance.Other ingredients are: ALC-0315(4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate),ALC-0159 (2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide),1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol,potassium chloride, potassium dihydrogen phosphate, sodium chloride,disodium phosphate dihydrate, sucrose and water for injection.

In some embodiments, a different buffer may be used in lieu of PBS. Insome embodiments, the buffer is formulated in a Tris-buffered solution.In some embodiments, the formulation comprises ALC-0315(4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate),ALC-0159 (2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide),DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), cholesterol, sucrose,trometamol (Tris), trometamol hydrochloride and water.

In some embodiments, the concentration of RNA in a pharmaceutical RNApreparation is about 0.1 mg/ml. In some embodiments about 30 ug of RNAis administered by administering about 200 uL of RNA preparation. Insome embodiments, RNA in a pharmaceutical RNA preparation is dilutedprior to administration (e.g., diluted to a concentration of about 0.05mg/ml). In some embodiments, administration volumes are between about200 μl and about 300 μl. In some embodiments, RNA in a pharmaceuticalRNA preparation is formulated in about 10 mM Tris buffer, and about 10%sucrose.

In some embodiments, the concentration of RNA in a pharmaceutical RNApreparation is about 0.1 mg/ml, and is formulated in about 10 mM Trisbuffer, about 10% sucrose and a dose of about 10 μg or RNA isadministered by diluting a pharmaceutical RNA preparation about 1:1 andadministering about 200 μl of diluted pharmaceutical RNA preparation. Insome embodiments, the concentration of RNA in a pharmaceutical RNApreparation is about 0.1 mg/ml, and is formulated in about 10 mM Trisbuffer, about 10% sucrose and a dose of RNA of about 10 μg isadministered by diluting a pharmaceutical RNA preparation about 1:5.75and administering about 200 μl of diluted pharmaceutical RNApreparation.

The amino acid sequence of the S protein encoded by BNT162b2 was chosenbased on the sequence for the “SARS-CoV-2 isolate Wuhan-Hu-1”: GenBank:MN908947.3 (complete genome) and GenBank: QHD43416.1 (spike surfaceglycoprotein). The BNT162b2 active substance consists of asingle-stranded, 5′-capped codon-optimized mRNA that is translated intothe spike antigen of SARS-CoV-2. The spike antigen protein sequenceencoded by BNT162b2 contains two proline mutations, which stabilizes anantigenically improved pre-fusion confirmation (P2 S). BNT162b2 does notcontain any uridines; instead of uridine the modifiedN1-methylpseudouridine is used in RNA synthesis. BNT162b2 mRNA istranslated into a SARS-CoV-2 S protein in host cells. The S protein isthen expressed on the cell surface where it induces an adaptive immuneresponse. The S protein encoded by BNT162b2 is identified as a targetfor neutralising antibodies against the virus and is considered arelevant vaccine component. For adult vaccine naïve subjects (i.e.,subjects 16 years and older who have not previously been administered aSARS-CoV-2 vaccine), the dosing regimen of BNT162b2 approved by the FDAcomprises administering intramuscularly (IM) two 30 μg doses of thediluted vaccine solution approximately 21 days apart.

The recent emergence of novel circulating variants of SARS-CoV-2 hasraised significant concerns about geographic and temporal efficacy ofvaccine interventions. One of the earliest variants that emerged andrapidly became globally dominant was D614G.

The alpha variant (also known as B.1.1.7, VOC202012/01, 501Y.V1 or GRY)was initially detected in the United Kingdom. The alpha variant has alarge number of mutations, including several mutations in the S gene. Ithas been shown to be inherently more transmissible, with a growth ratethat has been estimated to be 40-70% higher than other SARS-CoV-2lineages in multiple countries (Volz et al., 2021, Nature,https://doi.org/10.1038/s41586-021-03470-x; Washington et al., 2021,Cell https://doi.org/10.1016/j.cell.2021.03.052).

The beta variant (also known as B.1.351 or GH/501Y.V2) was firstdetected in South Africa. The beta variant carries several mutations inthe S gene. Three of these mutations are at sites in the RBD that areassociated with immune evasion: N501Y (shared with alpha) and E484K andK417N.

The gamma variant (also known as P.1 or GR/501Y.V3) was first detectedin Brazil. The gamma variant carries several mutations that affect thespike protein, including two shared with beta (N501Y and E484K), as wellas a different mutation at position 417 (K417T).

The delta variant (also known as B.1.617.2 or G/478K.V1) was firstdocumented in India. The delta variant has several point mutations thataffect the spike protein, including P681R (a mutation position sharedwith alpha and adjacent to the furin cleavage site), and L452R, which isin the RBD and has been linked with increased binding to ACE2 andneutralizing antibody resistance. There is also a deletion in the spikeprotein at position 156/157.

These four VOCs have circulated globally and became dominant variants inthe geographic regions where they were first identified.

On 24 Nov. 2021, the Omicron (B.1.1.529) variant was first reported toWHO from South Africa. SARS-CoV-2 Omicron and its sublineages have had amajor impact on the epidemiological landscape of the COVID-19 pandemicsince initial emergence in November 2021 (WHO Technical Advisory Groupon SARS-CoV-2 Virus Evolution (TAG-VE): Classification of Omicron(B.1.1.259): SARS-CoV-2 Variant of Concern (2021); WHO Headquarters(HQ), WHO Health Emergencies Programme, Enhancing Response to OmicronSARS-CoV-2 variant: Technical brief and priority actions for MemberStates (2022)). Significant alterations in the spike (S) glycoprotein ofthe first Omicron variant BA.1 leading to the loss of many neutralizingantibody epitopes (M. Hoffmann et al., “The Omicron variant is highlyresistant against antibody mediated neutralization: Implications forcontrol of the COVID-19 pandemic”, Cell 185, 447-456.e11 (2022))rendered BA.1 capable of partially escaping previously establishedSARS-CoV-2 wild-type strain (Wuhan-Hu-1)-based immunity (V. Servellita,et al., “Neutralizing immunity in vaccine breakthrough infections fromthe SARS-CoV-2 Omicron and Delta variants”, Cell 185, 1539-1548.e5(2022); Y. Cao et al., “Omicron escapes the majority of existingSARS-CoV-2 neutralizing antibodies”, Nature 602, 657-663 (2022)). Hence,breakthrough infection of vaccinated individuals with Omicron are morecommon than with previous Variants of Concern (VOCs). While Omicron BA.1was displaced by the BA.2 variant in many countries around the globe,other variants such as BA.1.1 and BA.3 temporarily and/or locally gainedmomentum but did not become globally dominant (S. Xia et al., “Origin,virological features, immune evasion and intervention of SARS-CoV-2Omicron sublineages. Signal Transduct. Target. Ther. 7, 241 (2022); H.Gruell et al., “SARS-CoV-2 Omicron sublineages exhibit distinct antibodyescape patterns, Cell Host Microbe 7, 241 (2022).). Omicron BA.2.12.1subsequently displaced BA.2 to become dominant in the United States,whereas BA.4 and BA.5 displaced BA.2 in Europe, parts of Africa, andAsia/Pacific (H. Gruell et al., “SARS-CoV-2 Omicron sublineages exhibitdistinct antibody escape patterns,” Cell Host Microbe 7, 241 (2022);European Centre for Disease Prevention and Control, Weekly COVID-19country overview—Country overview report: Week 31 2022 (2022); J.Hadfield et al., “Nextstrain: Real-time tracking of pathogen evolution,”Bioinformatics 34, 4121-4123 (2018)). Currently, Omicron BA.5 isdominant globally, including in the United States (Centers for DiseaseControl and Prevention. COVID Data Tracker. Atlanta, GA: US Departmentof Health and Human Services, CDC; 2022, August 12.https://covid.cdc.gov/coviddata-tracker (2022)). Omicron has acquirednumerous alterations (amino acid exchanges, insertions, or deletions) inthe S glycoprotein, among which some are shared between all Omicron VOCswhile others are specific to one or more Omicron sublineages.Antigenically, BA.2.12.1 exhibits high similarity with BA.2 but notBA.1, whereas BA.4 and BA.5 differ considerably from their ancestor BA.2and even more so from BA.1, in line with their genealogy (A. Z. Mykytynet al., “Antigenic cartography of SARS-CoV-2 reveals that Omicron BA.1and BA.2 are antigenically distinct,” Sci. Immunol. 7, eabg4450(2022).). Major differences of BA.1 from the remaining Omicron VOCsinclude Δ143-145, L212I, or ins214EPE in the S glycoprotein N-terminaldomain and G446S or G496S in the receptor binding domain (RBD). Aminoacid changes T376A, D405N, and R408S in the RBD are in turn common toBA.2 and its descendants but not found in BA.1. In addition, somealterations are specific for individual BA.2-descendant VOCs, includingL452Q for BA.2.12.1 or L452R and F486V for BA.4 and BA.5 (BA.4 and BA.5encode for the same S sequence). Most of these shared and VOC-specificalterations were shown to play an important role in immune escape frommonoclonal antibodies and polyclonal sera raised against the wild-type Sglycoprotein. In particular, the BA.4/BA.5-specific alterations arestrongly implicated in immune escape of these VOCs (P. Wang et al.,“Antibody resistance of SARS-CoV-2 variants B.1.351 and B.1.1.7. Nature593, 130-135 (2021); Q. Wang et al., “Antibody evasion by SARS-CoV-2Omicron subvariants BA.2.12.1, BA.4, & BA.5. Nature 608, 603-608(2022)).

SUMMARY

The present disclosure generally embraces immunotherapeutic treatment ofa subject comprising administration of RNA, e.g., vaccine RNA, encodingan amino acid sequence, e.g., a vaccine antigen, comprising a SARS-CoV-2S protein, an immunogenic variant thereof, or an immunogenic fragment ofthe SARS-CoV-2 S protein or the immunogenic variant thereof, i.e., anantigenic peptide or protein. Thus, vaccine antigen comprises an epitopeof SARS-CoV-2 S protein for inducing an immune response againstcoronavirus S protein, in particular SARS-CoV-2 S protein, in thesubject. RNA encoding vaccine antigen is administered to provide(following expression of the polynucleotide by appropriate target cells)antigen for induction, i.e., stimulation, priming and/or expansion, ofan immune response, e.g., antibodies and/or immune effector cells, whichis targeted to target antigen (coronavirus S protein, in particularSARS-CoV-2 S protein) or a procession product thereof. In oneembodiment, the immune response which is to be induced according to thepresent disclosure is a B cell-mediated immune response, i.e., anantibody-mediated immune response. Additionally or alternatively, in oneembodiment, the immune response which is to be induced according to thepresent disclosure is a T cell-mediated immune response. In oneembodiment, the immune response is an anti-coronavirus, in particularanti-SARS-CoV-2 immune response.

Vaccines described herein comprise as an active principlesingle-stranded RNA that may be translated into protein upon enteringcells of a recipient. In addition to wildtype or codon-optimizedsequences encoding an antigen sequence, RNA may contain one or morestructural elements optimized for maximal efficacy with respect tostability and translational efficiency (e.g., 5′ cap, 5′ UTR, 3′ UTR,poly(A)-tail, and combinations thereof). In one embodiment, RNA containsall of these elements. In one embodiment, a cap1 structure may beutilized as specific capping structure at the 5′-end of an RNA drugsubstance. In one embodiment, beta-S-ARCA(D1) (m₂ ^(7,2′-O)GppSpG) or m₂^(7,3′-O)Gppp(m₁ ^(2′-O))ApG may be utilized as specific cappingstructure at the 5′-end of an RNA drug substance. As 5′-UTR sequence,the 5′-UTR sequence of the human alpha-globin mRNA, optionally with anoptimized ‘Kozak sequence’ to increase translational efficiency (e.g.,SEQ ID NO: 12) may be used. As 3′-UTR sequence, a combination of twosequence elements (FI element) derived from the “amino terminal enhancerof split” (AES) mRNA (called F) and the mitochondrial encoded 125ribosomal RNA (called I) (e.g., SEQ ID NO: 13) placed between the codingsequence and the poly(A)-tail to assure higher maximum protein levelsand prolonged persistence of the mRNA may be used. These features wereidentified by an ex vivo selection process for sequences that confer RNAstability and augment total protein expression (see WO 2017/060314,herein incorporated by reference). Alternatively, the 3′-UTR may be twore-iterated 3′-UTRs of the human beta-globin mRNA. Additionally oralternatively, in some embodiments, a poly(A)-tail may comprise a lengthof at least 100 adenosine residues (including, e.g., at least 110adenosine residues, at least 120 adenosine residues, 130 adenosineresidues, or longer). In some embodiments, a poly(A)-tail may comprise alength of about 100 to about 150 adenosine residues. In some embodimentsa poly(A)-tail may comprise an interrupted poly(A)-tail. For example, insome such embodiments, a poly(A)-tail measuring 110 nucleotides inlength, consisting of a stretch of 30 adenosine residues, followed by a10 nucleotide linker sequence (of random nucleotides) and another 70adenosine residues (e.g., SEQ ID NO: 14) may be used. This poly(A)-tailsequence was designed to enhance RNA stability and translationalefficiency.

Furthermore, in some embodiments, a nucleotide sequence encoding asecretory signal peptide (sec) may be fused to antigen-encoding regionsof an RNA, preferably in some embodiments in a way that the sec istranslated as an N terminal tag. In one embodiment, sec corresponds tothe secretory signal peptide of a SARS-CoV-2 S protein (e.g., of a Wuhanstrain). In some embodiments, sequences coding for short linker peptidespredominantly consisting of the amino acids glycine (G) and serine (S),as commonly used for fusion proteins, may be used as GS/Linkers betweensec and an antigen.

Vaccine RNA described herein may be complexed with proteins and/orlipids, preferably lipids, to generate RNA-particles for administration.If a combination of different RNAs is used, RNAs may be complexedtogether or complexed separately with proteins and/or lipids to generateRNA-particles for administration.

In one aspect, the present disclosure relates to a composition ormedical preparation comprising RNA encoding an amino acid sequencecomprising a SARS-CoV-2 S protein, an immunogenic variant thereof, or animmunogenic fragment of the SARS-CoV-2 S protein or the immunogenicvariant thereof.

In one embodiment, an immunogenic fragment of the SARS-CoV-2 S proteincomprises the S1 subunit of the SARS-CoV-2 S protein, or the receptorbinding domain (RBD) of the S1 subunit of the SARS-CoV-2 S protein.

In one embodiment, an amino acid sequence comprising a SARS-CoV-2 Sprotein, an immunogenic variant thereof, or an immunogenic fragment ofthe SARS-CoV-2 S protein or the immunogenic variant thereof is able toform a multimeric complex, in particular a trimeric complex. To thisend, an amino acid sequence comprising a SARS-CoV-2 S protein, animmunogenic variant thereof, or an immunogenic fragment of theSARS-CoV-2 S protein or the immunogenic variant thereof may comprise adomain allowing the formation of a multimeric complex, in particular atrimeric complex of the amino acid sequence comprising a SARS-CoV-2 Sprotein, an immunogenic variant thereof, or an immunogenic fragment ofthe SARS-CoV-2 S protein or the immunogenic variant thereof. In oneembodiment, the domain allowing formation of a multimeric complexcomprises a trimerization domain, for example, a trimerization domain asdescribed herein, e.g., SARS-CoV-2 S protein trimerization domain. Inone embodiment, trimerization is achieved by addition of a trimerizationdomain, e.g., a T4-fibritin-derived “foldon” trimerization domain (e.g.,SEQ ID NO: 10), in particular if the amino acid sequence comprising aSARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenicfragment of the SARS-CoV-2 S protein or the immunogenic variant thereofcorresponds to a portion of a SARS-CoV-2 S protein that does notcomprise the SARS-CoV-2 S protein trimerization domain.

In one embodiment, the amino acid sequence comprising a SARS-CoV-2 Sprotein, an immunogenic variant thereof, or an immunogenic fragment ofthe SARS-CoV-2 S protein or the immunogenic variant thereof is encodedby a coding sequence which is codon-optimized and/or the G/C content ofwhich is increased compared to wild type coding sequence, wherein thecodon-optimization and/or the increase in the G/C content preferablydoes not change the sequence of the encoded amino acid sequence. Thoseskilled in the art will appreciate that codon optimization involveschoosing between or among alternative codons encoding the same aminoacid residue. Codon optimization typically includes consideration ofcodon(s) preferred by a particular host in which a sequence is to beexpressed. In accordance with the present disclosure, in manyembodiments, a preferred host is a human. In some embodiments, apreferred host may be a domestic animal. Alternatively or additionally,in some embodiments, selection between or among possible codons encodingthe same amino acid may consider one or more other features such as, forexample, overall G/C content (as noted above) and/or similarity to aparticular reference. For example, in some embodiments of the presentdisclosure, a provided coding sequence that encodes a SARS-CoV-2 Sprotein or immunogenic variant thereof that differs in amino acidsequence from that encoded by a BNT162b2 construct described hereinutilizes a codon, in at least one position of such difference, thatpreserves greater similarity to the BNT162b2 construct sequence relativeto at least one alternative codon encoding the same amino acid at suchposition of difference.

In one embodiment,

-   -   (i) the RNA encoding a SARS-CoV-2 S protein, an immunogenic        variant thereof, or an immunogenic fragment of the SARS-CoV-2 S        protein or the immunogenic variant thereof comprises the        nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2,        8 or 9, a nucleotide sequence having at least 99%, 98%, 97%,        96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence        of nucleotides 979 to 1584 of SEQ ID NO: 2, 8 or 9, or a        fragment of the nucleotide sequence of nucleotides 979 to 1584        of SEQ ID NO: 2, 8 or 9, or the nucleotide sequence having at        least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the        nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2,        8 or 9; and/or    -   (ii) a SARS-CoV-2 S protein, an immunogenic variant thereof, or        an immunogenic fragment of the SARS-CoV-2 S protein or the        immunogenic variant thereof comprises the amino acid sequence of        amino acids 327 to 528 of SEQ ID NO: 1, an amino acid sequence        having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%        identity to the amino acid sequence of amino acids 327 to 528 of        SEQ ID NO: 1, or an immunogenic fragment of the amino acid        sequence of amino acids 327 to 528 of SEQ ID NO: 1, or the amino        acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,        or 80% identity to the amino acid sequence of amino acids 327 to        528 of SEQ ID NO: 1.

In one embodiment,

-   -   (i) the RNA encoding a SARS-CoV-2 S protein, an immunogenic        variant thereof, or an immunogenic fragment of the SARS-CoV-2 S        protein or the immunogenic variant thereof comprises the        nucleotide sequence of nucleotides 111 to 986 of SEQ ID NO: 30,        a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%,        90%, 85%, or 80% identity to the nucleotide sequence of        nucleotides 111 to 986 of SEQ ID NO: 30, or a fragment of the        nucleotide sequence of nucleotides 111 to 986 of SEQ ID NO: 30,        or the nucleotide sequence having at least 99%, 98%, 97%, 96%,        95%, 90%, 85%, or 80% identity to the nucleotide sequence of        nucleotides 111 to 986 of SEQ ID NO: 30; and/or    -   (ii) a SARS-CoV-2 S protein, an immunogenic variant thereof, or        an immunogenic fragment of the SARS-CoV-2 S protein or the        immunogenic variant thereof comprises the amino acid sequence of        amino acids 20 to 311 of SEQ ID NO: 29, an amino acid sequence        having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%        identity to the amino acid sequence of amino acids 20 to 311 of        SEQ ID NO: 29, or an immunogenic fragment of the amino acid        sequence of amino acids 20 to 311 of SEQ ID NO: 29, or the amino        acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,        or 80% identity to the amino acid sequence of amino acids 20 to        311 of SEQ ID NO: 29.

In one embodiment,

-   -   (i) the RNA encoding a SARS-CoV-2 S protein, an immunogenic        variant thereof, or an immunogenic fragment of the SARS-CoV-2 S        protein or the immunogenic variant thereof comprises the        nucleotide sequence of nucleotides 49 to 2055 of SEQ ID NO: 2, 8        or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%,        95%, 90%, 85%, or 80% identity to the nucleotide sequence of        nucleotides 49 to 2055 of SEQ ID NO: 2, 8 or 9, or a fragment of        the nucleotide sequence of nucleotides 49 to 2055 of SEQ ID NO:        2, 8 or 9, or the nucleotide sequence having at least 99%, 98%,        97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide        sequence of nucleotides 49 to 2055 of SEQ ID NO: 2, 8 or 9;        and/or    -   (ii) a SARS-CoV-2 S protein, an immunogenic variant thereof, or        an immunogenic fragment of the SARS-CoV-2 S protein or the        immunogenic variant thereof comprises the amino acid sequence of        amino acids 17 to 685 of SEQ ID NO: 1, an amino acid sequence        having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%        identity to the amino acid sequence of amino acids 17 to 685 of        SEQ ID NO: 1, or an immunogenic fragment of the amino acid        sequence of amino acids 17 to 685 of SEQ ID NO: 1, or the amino        acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,        or 80% identity to the amino acid sequence of amino acids 17 to        685 of SEQ ID NO: 1.

In one embodiment,

-   -   (i) the RNA encoding a SARS-CoV-2 S protein, an immunogenic        variant thereof, or an immunogenic fragment of the SARS-CoV-2 S        protein or the immunogenic variant thereof comprises the        nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8        or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%,        95%, 90%, 85%, or 80% identity to the nucleotide sequence of        nucleotides 49 to 3819 of SEQ ID NO: 2, 8 or 9, or a fragment of        the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO:        2, 8 or 9, or the nucleotide sequence having at least 99%, 98%,        97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide        sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8 or 9;        and/or    -   (ii) a SARS-CoV-2 S protein, an immunogenic variant thereof, or        an immunogenic fragment of the SARS-CoV-2 S protein or the        immunogenic variant thereof comprises the amino acid sequence of        amino acids 17 to 1273 of SEQ ID NO: 1 or 7, an amino acid        sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or        80% identity to the amino acid sequence of amino acids 17 to        1273 of SEQ ID NO: 1 or 7, or an immunogenic fragment of the        amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or        7, or the amino acid sequence having at least 99%, 98%, 97%,        96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence        of amino acids 17 to 1273 of SEQ ID NO: 1 or 7.

In one embodiment, the amino acid sequence comprising a SARS-CoV-2 Sprotein, an immunogenic variant thereof, or an immunogenic fragment ofthe SARS-CoV-2 S protein or the immunogenic variant thereof comprises asecretory signal peptide.

In one embodiment, the secretory signal peptide is fused, preferablyN-terminally, to a SARS-CoV-2 S protein, an immunogenic variant thereof,or an immunogenic fragment of the SARS-CoV-2 S protein or theimmunogenic variant thereof.

In one embodiment,

-   -   (i) the RNA encoding the secretory signal peptide comprises the        nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8 or        9, a nucleotide sequence having at least 99%, 98%, 97%, 96%,        95%, 90%, 85%, or 80% identity to the nucleotide sequence of        nucleotides 1 to 48 of SEQ ID NO: 2, 8 or 9, or a fragment of        the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2,        8 or 9, or the nucleotide sequence having at least 99%, 98%,        97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide        sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8 or 9; and/or    -   (ii) the secretory signal peptide comprises the amino acid        sequence of amino acids 1 to 16 of SEQ ID NO: 1, an amino acid        sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or        80% identity to the amino acid sequence of amino acids 1 to 16        of SEQ ID NO: 1, or a functional fragment of the amino acid        sequence of amino acids 1 to 16 of SEQ ID NO: 1, or the amino        acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,        or 80% identity to the amino acid sequence of amino acids 1 to        16 of SEQ ID NO: 1.

In one embodiment,

-   -   (i) the RNA encoding a SARS-CoV-2 S protein, an immunogenic        variant thereof, or an immunogenic fragment of the SARS-CoV-2 S        protein or the immunogenic variant thereof comprises the        nucleotide sequence of SEQ ID NO: 6, a nucleotide sequence        having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%        identity to the nucleotide sequence of SEQ ID NO: 6, or a        fragment of the nucleotide sequence of SEQ ID NO: 6, or the        nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%,        90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID        NO: 6; and/or    -   (ii) a SARS-CoV-2 S protein, an immunogenic variant thereof, or        an immunogenic fragment of the SARS-CoV-2 S protein or the        immunogenic variant thereof comprises the amino acid sequence of        SEQ ID NO: 5, an amino acid sequence having at least 99%, 98%,        97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid        sequence of SEQ ID NO: 5, or an immunogenic fragment of the        amino acid sequence of SEQ ID NO: 5, or the amino acid sequence        having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%        identity to the amino acid sequence of SEQ ID NO: 5.

In one embodiment,

-   -   (i) the RNA encoding a SARS-CoV-2 S protein, an immunogenic        variant thereof, or an immunogenic fragment of the SARS-CoV-2 S        protein or the immunogenic variant thereof comprises the        nucleotide sequence of nucleotides 54 to 986 of SEQ ID NO: 30, a        nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%,        90%, 85%, or 80% identity to the nucleotide sequence of        nucleotides 54 to 986 of SEQ ID NO: 30, or a fragment of the        nucleotide sequence of nucleotides 54 to 986 of SEQ ID NO: 30,        or the nucleotide sequence having at least 99%, 98%, 97%, 96%,        95%, 90%, 85%, or 80% identity to the nucleotide sequence of        nucleotides 54 to 986 of SEQ ID NO: 30; and/or    -   (ii) a SARS-CoV-2 S protein, an immunogenic variant thereof, or        an immunogenic fragment of the SARS-CoV-2 S protein or the        immunogenic variant thereof comprises the amino acid sequence of        amino acids 1 to 311 of SEQ ID NO: 29, an amino acid sequence        having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%        identity to the amino acid sequence of amino acids 1 to 311 of        SEQ ID NO: 29, or an immunogenic fragment of the amino acid        sequence of amino acids 1 to 311 of SEQ ID NO: 29, or the amino        acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,        or 80% identity to the amino acid sequence of amino acids 1 to        311 of SEQ ID NO: 29.

In one embodiment, the RNA is a modified RNA, in particular a stabilizedmRNA. In one embodiment, the RNA comprises a modified nucleoside inplace of at least one uridine. In one embodiment, the RNA comprises amodified nucleoside in place of each uridine. In one embodiment, themodified nucleoside is independently selected from pseudouridine (ψ),N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U).

In one embodiment, RNA comprises a modified nucleoside in place ofuridine.

In one embodiment, the modified nucleoside is selected frompseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine(m5U).

In one embodiment, RNA comprises a 5′ cap.

In one embodiment, RNA encoding an amino acid sequence comprising aSARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenicfragment of the SARS-CoV-2 S protein or the immunogenic variant thereofcomprises a 5′ UTR comprising the nucleotide sequence of SEQ ID NO: 12,or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%,85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 12.

In one embodiment, RNA encoding an amino acid sequence comprising aSARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenicfragment of the SARS-CoV-2 S protein or the immunogenic variant thereofcomprises a 3′ UTR comprising the nucleotide sequence of SEQ ID NO: 13,or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%,85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 13.

In one embodiment, RNA encoding an amino acid sequence comprising aSARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenicfragment of the SARS-CoV-2 S protein or the immunogenic variant thereofcomprises a poly-A sequence.

In one embodiment, the poly-A sequence comprises at least 100nucleotides.

In one embodiment, the poly-A sequence comprises or consists of thenucleotide sequence of SEQ ID NO: 14.

In one embodiment, RNA is formulated or is to be formulated as a liquid,a solid, or a combination thereof.

In one embodiment, RNA is formulated or is to be formulated forinjection.

In one embodiment, RNA is formulated or is to be formulated forintramuscular administration.

In one embodiment, RNA is formulated or is to be formulated asparticles.

In one embodiment, particles are lipid nanoparticles (LNP) or lipoplex(LPX) particles.

In one embodiment, LNPs comprise((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate),2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide,1,2-Distearoyl-sn-glycero-3-phosphocholine, and cholesterol.

In one embodiment, RNA lipoplex particles are obtainable by mixing RNAwith liposomes. In one embodiment, RNA lipoplex particles are obtainableby mixing RNA with lipids.

In one embodiment, RNA is formulated or is to be formulated as colloid.In one embodiment, RNA is formulated or is to be formulated asparticles, forming the dispersed phase of a colloid.

In one embodiment, 50% or more, 75% or more, or 85% or more of RNA ispresent in the dispersed phase. In one embodiment, RNA is formulated oris to be formulated as particles comprising RNA and lipids. In oneembodiment, particles are formed by exposing RNA, dissolved in anaqueous phase, with lipids, dissolved in an organic phase. In oneembodiment, the organic phase comprises ethanol. In one embodiment,particles are formed by exposing RNA, dissolved in an aqueous phase,with lipids, dispersed in an aqueous phase. In one embodiment, thelipids dispersed in an aqueous phase form liposomes.

In one embodiment, RNA is mRNA or saRNA.

In one embodiment, a composition or medical preparation is apharmaceutical composition.

In one embodiment, a composition or medical preparation is a vaccine.

In one embodiment, a pharmaceutical composition further comprises one ormore pharmaceutically acceptable carriers, diluents and/or excipients.

In one embodiment, a composition or medical preparation is a kit.

In one embodiment, RNA and optionally the particle forming componentsare in separate vials.

In one embodiment, a kit further comprises instructions for use of acomposition or medical preparation for inducing an immune responseagainst coronavirus in a subject.

In one aspect, the present disclosure relates to a composition ormedical preparation described herein for pharmaceutical use.

In one embodiment, a pharmaceutical use comprises inducing an immuneresponse against coronavirus in a subject.

In one embodiment, a pharmaceutical use comprises a therapeutic orprophylactic treatment of a coronavirus infection.

In one embodiment, a composition or medical preparation described hereinis for administration to a human.

In one embodiment, the coronavirus is a betacoronavirus.

In one embodiment, the coronavirus is a sarbecovirus.

In one embodiment, the coronavirus is SARS-CoV-2.

In one aspect, the present disclosure relates to a method of inducing animmune response against coronavirus in a subject comprisingadministering to the subject a composition comprising RNA encoding anamino acid sequence comprising a SARS-CoV-2 S protein, an immunogenicvariant thereof, or an immunogenic fragment of the SARS-CoV-2 S proteinor the immunogenic variant thereof.

In one embodiment, an immunogenic fragment of the SARS-CoV-2 S proteincomprises the S1 subunit of the SARS-CoV-2 S protein, or the receptorbinding domain (RBD) of the S1 subunit of the SARS-CoV-2 S protein.

In one embodiment, an amino acid sequence comprising a SARS-CoV-2 Sprotein, an immunogenic variant thereof, or an immunogenic fragment ofthe SARS-CoV-2 S protein or the immunogenic variant thereof is able toform a multimeric complex, in particular a trimeric complex. To thisend, an amino acid sequence comprising a SARS-CoV-2 S protein, animmunogenic variant thereof, or an immunogenic fragment of theSARS-CoV-2 S protein or the immunogenic variant thereof may comprise adomain allowing the formation of a multimeric complex, in particular atrimeric complex of the amino acid sequence comprising a SARS-CoV-2 Sprotein, an immunogenic variant thereof, or an immunogenic fragment ofthe SARS-CoV-2 S protein or the immunogenic variant thereof. In oneembodiment, the domain allowing the formation of a multimeric complexcomprises a trimerization domain, for example, a trimerization domain asdescribed herein.

In one embodiment, an amino acid sequence comprising a SARS-CoV-2 Sprotein, an immunogenic variant thereof, or an immunogenic fragment ofthe SARS-CoV-2 S protein or the immunogenic variant thereof is encodedby a coding sequence which is codon-optimized and/or the G/C content ofwhich is increased compared to wild type coding sequence, wherein thecodon-optimization and/or the increase in the G/C content preferablydoes not change the sequence of the encoded amino acid sequence.

In one embodiment,

-   -   (i) the RNA encoding a SARS-CoV-2 S protein, an immunogenic        variant thereof, or an immunogenic fragment of the SARS-CoV-2 S        protein or the immunogenic variant thereof comprises the        nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2,        8 or 9, a nucleotide sequence having at least 99%, 98%, 97%,        96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence        of nucleotides 979 to 1584 of SEQ ID NO: 2, 8 or 9, or a        fragment of the nucleotide sequence of nucleotides 979 to 1584        of SEQ ID NO: 2, 8 or 9, or the nucleotide sequence having at        least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the        nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2,        8 or 9; and/or    -   (ii) a SARS-CoV-2 S protein, an immunogenic variant thereof, or        an immunogenic fragment of the SARS-CoV-2 S protein or the        immunogenic variant thereof comprises the amino acid sequence of        amino acids 327 to 528 of SEQ ID NO: 1, an amino acid sequence        having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%        identity to the amino acid sequence of amino acids 327 to 528 of        SEQ ID NO: 1, or an immunogenic fragment of the amino acid        sequence of amino acids 327 to 528 of SEQ ID NO: 1, or the amino        acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,        or 80% identity to the amino acid sequence of amino acids 327 to        528 of SEQ ID NO: 1.

In one embodiment,

-   -   (i) the RNA encoding a SARS-CoV-2 S protein, an immunogenic        variant thereof, or an immunogenic fragment of the SARS-CoV-2 S        protein or the immunogenic variant thereof comprises the        nucleotide sequence of nucleotides 111 to 986 of SEQ ID NO: 30,        a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%,        90%, 85%, or 80% identity to the nucleotide sequence of        nucleotides 111 to 986 of SEQ ID NO: 30, or a fragment of the        nucleotide sequence of nucleotides 111 to 986 of SEQ ID NO: 30,        or the nucleotide sequence having at least 99%, 98%, 97%, 96%,        95%, 90%, 85%, or 80% identity to the nucleotide sequence of        nucleotides 111 to 986 of SEQ ID NO: 30; and/or    -   (ii) a SARS-CoV-2 S protein, an immunogenic variant thereof, or        an immunogenic fragment of the SARS-CoV-2 S protein or the        immunogenic variant thereof comprises the amino acid sequence of        amino acids 20 to 311 of SEQ ID NO: 29, an amino acid sequence        having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%        identity to the amino acid sequence of amino acids 20 to 311 of        SEQ ID NO: 29, or an immunogenic fragment of the amino acid        sequence of amino acids 20 to 311 of SEQ ID NO: 29, or the amino        acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,        or 80% identity to the amino acid sequence of amino acids 20 to        311 of SEQ ID NO: 29.

In one embodiment,

-   -   (i) the RNA encoding a SARS-CoV-2 S protein, an immunogenic        variant thereof, or an immunogenic fragment of the SARS-CoV-2 S        protein or the immunogenic variant thereof comprises the        nucleotide sequence of nucleotides 49 to 2055 of SEQ ID NO: 2, 8        or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%,        95%, 90%, 85%, or 80% identity to the nucleotide sequence of        nucleotides 49 to 2055 of SEQ ID NO: 2, 8 or 9, or a fragment of        the nucleotide sequence of nucleotides 49 to 2055 of SEQ ID NO:        2, 8 or 9, or the nucleotide sequence having at least 99%, 98%,        97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide        sequence of nucleotides 49 to 2055 of SEQ ID NO: 2, 8 or 9;        and/or    -   (ii) a SARS-CoV-2 S protein, an immunogenic variant thereof, or        an immunogenic fragment of the SARS-CoV-2 S protein or the        immunogenic variant thereof comprises the amino acid sequence of        amino acids 17 to 685 of SEQ ID NO: 1, an amino acid sequence        having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%        identity to the amino acid sequence of amino acids 17 to 685 of        SEQ ID NO: 1, or an immunogenic fragment of the amino acid        sequence of amino acids 17 to 685 of SEQ ID NO: 1, or the amino        acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,        or 80% identity to the amino acid sequence of amino acids 17 to        685 of SEQ ID NO: 1.

In one embodiment,

-   -   (i) the RNA encoding a SARS-CoV-2 S protein, an immunogenic        variant thereof, or an immunogenic fragment of the SARS-CoV-2 S        protein or the immunogenic variant thereof comprises the        nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8        or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%,        95%, 90%, 85%, or 80% identity to the nucleotide sequence of        nucleotides 49 to 3819 of SEQ ID NO: 2, 8 or 9, or a fragment of        the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO:        2, 8 or 9, or the nucleotide sequence having at least 99%, 98%,        97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide        sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8 or 9;        and/or    -   (ii) a SARS-CoV-2 S protein, an immunogenic variant thereof, or        an immunogenic fragment of the SARS-CoV-2 S protein or the        immunogenic variant thereof comprises the amino acid sequence of        amino acids 17 to 1273 of SEQ ID NO: 1 or 7, an amino acid        sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or        80% identity to the amino acid sequence of amino acids 17 to        1273 of SEQ ID NO: 1 or 7, or an immunogenic fragment of the        amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or        7, or the amino acid sequence having at least 99%, 98%, 97%,        96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence        of amino acids 17 to 1273 of SEQ ID NO: 1 or 7.

In one embodiment, an amino acid sequence comprising a SARS-CoV-2 Sprotein, an immunogenic variant thereof, or an immunogenic fragment ofthe SARS-CoV-2 S protein or the immunogenic variant thereof comprises asecretory signal peptide.

In one embodiment, a secretory signal peptide is fused, preferablyN-terminally, to a SARS-CoV-2 S protein, an immunogenic variant thereof,or an immunogenic fragment of the SARS-CoV-2 S protein or theimmunogenic variant thereof.

In one embodiment,

-   -   (i) the RNA encoding the secretory signal peptide comprises the        nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8 or        9, a nucleotide sequence having at least 99%, 98%, 97%, 96%,        95%, 90%, 85%, or 80% identity to the nucleotide sequence of        nucleotides 1 to 48 of SEQ ID NO: 2, 8 or 9, or a fragment of        the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2,        8 or 9, or the nucleotide sequence having at least 99%, 98%,        97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide        sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8 or 9; and/or    -   (ii) the secretory signal peptide comprises the amino acid        sequence of amino acids 1 to 16 of SEQ ID NO: 1, an amino acid        sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or        80% identity to the amino acid sequence of amino acids 1 to 16        of SEQ ID NO: 1, or a functional fragment of the amino acid        sequence of amino acids 1 to 16 of SEQ ID NO: 1, or the amino        acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,        or 80% identity to the amino acid sequence of amino acids 1 to        16 of SEQ ID NO: 1.

In one embodiment,

-   -   (i) the RNA encoding a SARS-CoV-2 S protein, an immunogenic        variant thereof, or an immunogenic fragment of the SARS-CoV-2 S        protein or the immunogenic variant thereof comprises the        nucleotide sequence of SEQ ID NO: 6, a nucleotide sequence        having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%        identity to the nucleotide sequence of SEQ ID NO: 6, or a        fragment of the nucleotide sequence of SEQ ID NO: 6, or the        nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%,        90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID        NO: 6; and/or    -   (ii) a SARS-CoV-2 S protein, an immunogenic variant thereof, or        an immunogenic fragment of the SARS-CoV-2 S protein or the        immunogenic variant thereof comprises the amino acid sequence of        SEQ ID NO: 5, an amino acid sequence having at least 99%, 98%,        97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid        sequence of SEQ ID NO: 5, or an immunogenic fragment of the        amino acid sequence of SEQ ID NO: 5, or the amino acid sequence        having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%        identity to the amino acid sequence of SEQ ID NO: 5.

In one embodiment,

-   -   (i) the RNA encoding a SARS-CoV-2 S protein, an immunogenic        variant thereof, or an immunogenic fragment of the SARS-CoV-2 S        protein or the immunogenic variant thereof comprises the        nucleotide sequence of nucleotides 54 to 986 of SEQ ID NO: 30, a        nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%,        90%, 85%, or 80% identity to the nucleotide sequence of        nucleotides 54 to 986 of SEQ ID NO: 30, or a fragment of the        nucleotide sequence of nucleotides 54 to 986 of SEQ ID NO: 30,        or the nucleotide sequence having at least 99%, 98%, 97%, 96%,        95%, 90%, 85%, or 80% identity to the nucleotide sequence of        nucleotides 54 to 986 of SEQ ID NO: 30; and/or    -   (ii) a SARS-CoV-2 S protein, an immunogenic variant thereof, or        an immunogenic fragment of the SARS-CoV-2 S protein or the        immunogenic variant thereof comprises the amino acid sequence of        amino acids 1 to 311 of SEQ ID NO: 29, an amino acid sequence        having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%        identity to the amino acid sequence of amino acids 1 to 311 of        SEQ ID NO: 29, or an immunogenic fragment of the amino acid        sequence of amino acids 1 to 311 of SEQ ID NO: 29, or the amino        acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,        or 80% identity to the amino acid sequence of amino acids 1 to        311 of SEQ ID NO: 29.

In one embodiment, RNA is modified RNA, in particular stabilized mRNA.In one embodiment, RNA comprises a modified nucleoside in place of atleast one uridine. In one embodiment, RNA comprises a modifiednucleoside in place of each uridine. In one embodiment, a modifiednucleoside is independently selected from pseudouridine (ψ),N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U).

In one embodiment, RNA comprises a modified nucleoside in place ofuridine.

In one embodiment, the modified nucleoside is selected frompseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine(m5U).

In one embodiment, RNA comprises a cap.

In one embodiment, RNA encoding an amino acid sequence comprising aSARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenicfragment of the SARS-CoV-2 S protein or the immunogenic variant thereofcomprises a 5′ UTR comprising the nucleotide sequence of SEQ ID NO: 12,or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%,85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 12.

In one embodiment, RNA encoding an amino acid sequence comprising aSARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenicfragment of the SARS-CoV-2 S protein or the immunogenic variant thereofcomprises a 3′ UTR comprising the nucleotide sequence of SEQ ID NO: 13,or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%,85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 13.

In one embodiment, RNA encoding an amino acid sequence comprising aSARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenicfragment of the SARS-CoV-2 S protein or the immunogenic variant thereofcomprises a poly-A sequence.

In one embodiment, a poly-A sequence comprises at least 100 nucleotides.

In one embodiment, a poly-A sequence comprises or consists of thenucleotide sequence of SEQ ID NO: 14.

In one embodiment, RNA is formulated as a liquid, a solid, or acombination thereof.

In one embodiment, RNA is administered by injection.

In one embodiment, RNA is administered by intramuscular administration.

In one embodiment, RNA is formulated as particles.

In one embodiment, the particles are lipid nanoparticles (LNP) orlipoplex (LPX) particles.

In one embodiment, LNPs comprise((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate),2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide,1,2-Distearoyl-sn-glycero-3-phosphocholine, and cholesterol.

In one embodiment, RNA lipoplex particles are obtainable by mixing RNAwith liposomes. In one embodiment, RNA lipoplex particles are obtainableby mixing RNA with lipids.

In one embodiment, RNA is formulated as colloid. In one embodiment, RNAis formulated as particles, forming the dispersed phase of a colloid. Inone embodiment, 50% or more, 75% or more, or 85% or more of RNA arepresent in the dispersed phase. In one embodiment, RNA is formulated asparticles comprising RNA and lipids. In one embodiment, particles areformed by exposing RNA, dissolved in an aqueous phase, with lipids,dissolved in an organic phase. In one embodiment, the organic phasecomprises ethanol. In one embodiment, particles are formed by exposingRNA, dissolved in an aqueous phase, with lipids, dispersed in an aqueousphase. In one embodiment, the lipids dispersed in an aqueous phase formliposomes.

In one embodiment, RNA is mRNA or saRNA.

In one embodiment, a method disclosed herein is a method for vaccinationagainst coronavirus.

In one embodiment, a method disclosed herein is a method for therapeuticor prophylactic treatment of a coronavirus infection.

In one embodiment, a subject is a human.

In one embodiment, the coronavirus is a betacoronavirus.

In one embodiment, the coronavirus is a sarbecovirus.

In one embodiment, the coronavirus is SARS-CoV-2.

In one embodiment of methods described herein, a composition describedherein is administered to a subject.

In one aspect, the present disclosure relates to a composition ormedical preparation described herein for use in a method describedherein.

Among other things, the present disclosure teaches that a compositioncomprising a lipid nanoparticle encapsulated RNA encoding at least aportion (e.g., that is or comprises an epitope) of a SARS-CoV-2-encodedpolypeptide (e.g., of a SARS-CoV-2-encoded S protein) can achievedetectable antibody titer against an epitope in serum within 7 daysafter administration to a population of adult human subjects accordingto a regimen that includes administration of at least one dose of thecomposition. Moreover, the present disclosure teaches persistence ofsuch antibody titer. In some embodiments, such antibody titer isincreased when a modified mRNA is used, as compared with titer achievedwith a corresponding unmodified mRNA.

In some embodiments, a provided regimen includes at least one dose. Insome embodiments, a provided regimen includes a first dose and at leastone subsequent dose. In some embodiments, the first dose is the sameamount as at least one subsequent dose. In some embodiments, the firstdose is the same amount as all subsequent doses. In some embodiments,the first dose is a different amount as at least one subsequent dose. Insome embodiments, the first dose is a different amount than allsubsequent doses. In some embodiments, a provided regimen comprises twodoses. In some embodiments, a provided regimen consists of two doses.

In particular embodiments, an immunogenic composition is formulated as asingle-dose in a container, e.g., a vial. In some embodiments, animmunogenic composition is formulated as a multi-dose formulation in avial. In some embodiments, the multi-dose formulation includes at least2 doses per vial. In some embodiments, the multi-dose formulationincludes a total of 2-20 doses per vial, such as, for example, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, or 12 doses per vial. In some embodiments, eachdose in the vial is equal in volume. In some embodiments, a first doseis a different volume than a subsequent dose.

A “stable” multi-dose formulation exhibits no unacceptable levels ofmicrobial growth, and substantially no or no breakdown or degradation ofthe active biological molecule component(s). As used herein, a “stable”immunogenic composition includes a formulation that remains capable ofeliciting a desired immunologic response when administered to a subject.

In some embodiments, a multi-dose formulation remains stable for aspecified time with multiple or repeated inoculations/insertions intothe multi-dose container. For example, in some embodiments a multi-doseformulation may be stable for at least three days with up to ten usages,when contained within a multi-dose container. In some embodiments,multi-dose formulations remain stable with 2-20 inoculations/insertions.

In some embodiments, administration of a composition comprising a lipidnanoparticle encapsulated RNA (e.g., in some embodiments mRNA) encodingat least a portion (e.g., that is or comprises an epitope) of aSARS-CoV-2-encoded polypeptide (e.g., of a SARS-CoV-2-encoded Sprotein), e.g., according to a regimen as described herein, may resultin lymphopenia in some subjects (e.g., in all subjects, in mostsubjects, in about 50% or fewer, in about 40% or fewer, in about 40% orfewer, in about 25% or fewer, in about 20% or fewer, in about 15% orfewer, in about 10% or fewer, in about 5% or fewer, etc). Among otherthings, the present disclosure teaches that such lymphopenia can resolveover time. For example, in some embodiments, lymphopenia resolves withinabout 14, about 10, about 9, about 8, about 7 days or less. In someembodiments, lymphopenia is Grade 3, Grade 2, or less.

Thus, among other things, the present disclosure provides compositionscomprising a lipid nanoparticle encapsulated RNA (e.g., in someembodiments mRNA) encoding at least a portion (e.g., that is orcomprises an epitope) of a SARS-CoV-2-encoded polypeptide (e.g., of aSARS-CoV-2-encoded S protein) that are characterized, when administeredto a relevant population of adults, to display certain characteristics(e.g., achieve certain effects) as described herein. In someembodiments, provided compositions may have been prepared, stored,transported, characterized, and/or used under conditions wheretemperature does not exceed a particular threshold. Alternatively oradditionally, in some embodiments, provided compositions may have beenprotected from light (e.g., from certain wavelengths) during some or allof their preparation, storage, transport, characterization, and/or use.In some embodiments, one or more features of provided compositions(e.g., RNA stability, as may be assessed, for example, by one or more ofsize, presence of particular moiety or modification, etc; lipidnanoparticle stability or aggregation, pH, etc) may be or have beenassessed at one or more points during preparation, storage, transport,and/or use prior to administration.

Among other things, the present disclosure documents that certainprovided compositions in which nucleotides within an RNA (e.g., in someembodiments mRNA) are not modified (e.g., are naturally-occurring A, U,C, G), and/or provided methods relating to such compositions, arecharacterized (e.g., when administered to a relevant population, whichmay in some embodiments be or comprise an adult population), by anintrinsic adjuvant effect. In some embodiments, such a compositionand/or method can induce an antibody and/or a T cell response. In someembodiments, such a composition and/or method can induce a higher T cellresponse, as compared to conventional vaccines (e.g., non-RNA vaccinessuch as protein vaccines).

Alternatively or additionally, the present disclosure documents thatprovided compositions (e.g., compositions comprising a lipidnanoparticle encapsulated RNA encoding at least a portion (e.g., that isor comprises an epitope) of a SARS-CoV-2-encoded polypeptide (e.g., of aSARS-CoV-2-encoded S protein)) in which nucleotides within an RNA aremodified, and/or provided methods relating to such compositions, arecharacterized (e.g., when administered to a relevant population, whichmay in some embodiments be or comprise an adult population), by absenceof an intrinsic adjuvant effect, or by a reduced intrinsic adjuvanteffect as compared with an otherwise comparable composition (or method)with unmodified results. Alternatively or additionally, in someembodiments, such compositions (or methods) are characterized in thatthey (e.g., when administered to a relevant population, which may insome embodiments be or comprise an adult population) induce an antibodyresponse and/or a CD4+ T cell response. Still further alternatively oradditionally, in some embodiments, such compositions (or methods) arecharacterized in that they (e.g., when administered to a relevantpopulation, which may in some embodiments be or comprise an adultpopulation) induce a higher CD4+ T cell response than that observed withan alternative vaccine format (e.g., a peptide vaccine). In someembodiments involving modified nucleotides, such modified nucleotidesmay be present, for example, in a 3′ UTR sequence, an antigen-encodingsequence, and/or a 5′UTR sequence. In some embodiments, modifiednucleotides are or include one or more modified uracil residues and/orone or more modified cytosine residues. Among other things, the presentdisclosure documents that provided (e.g., compositions comprising alipid nanoparticle encapsulated RNA encoding at least a portion (e.g.,that is or comprises an epitope) of a SARS-CoV-2-encoded polypeptide(e.g., of a SARS-CoV-2-encoded S protein)) and/or methods arecharacterized by (e.g., when administered to a relevant population,which may in some embodiments be or comprise an adult population)sustained expression of an encoded polypeptide (e.g., of aSARS-CoV-2-encoded protein [such as an S protein] or portion thereof,which portion, in some embodiments, may be or comprise an epitopethereof). For example, in some embodiments, such compositions and/ormethods are characterized in that, when administered to a human, theyachieve detectable polypeptide expression in a biological sample (e.g.,serum) from such human and, in some embodiments, such expressionpersists for a period of time that is at least at least 36 hours orlonger, including, e.g., at least 48 hours, at least 60 hours, at least72 hours, at least 96 hours, at least 120 hours, at least 148 hours, orlonger.

Those skilled in the art, reading the present disclosure, willappreciate that it describes various RNA constructs (e.g., in someembodiments mRNA constructs) encoding at least a portion (e.g., that isor comprises an epitope) of a SARS-CoV-2-encoded polypeptide (e.g., of aSARS-CoV-2-encoded S protein)). Such person of ordinary skill, readingthe present disclosure, will particularly appreciate that it describesvarious RNA constructs (e.g., in some embodiments mRNA constructs)encoding at least a portion of a SARS-CoV-2 S protein, for example atleast an RBD portion of a SARS-CoV-2 S protein. Still further, such aperson of ordinary skill, reading the present disclosure, willappreciate that it describes particular characteristics and/oradvantages of RNA constructs (e.g., in some embodiments mRNA constructs)encoding at least a portion (e.g., that is or comprises an epitope) of aSARS-CoV-2-encoded polypeptide (e.g., of a SARS-CoV-2-encoded Sprotein). In some embodiments, an RNA construct (e.g., in someembodiments, an mRNA construct) may encode at least one domain of aSARS-CoV-2 encoded polypeptide (e.g., one or more domains of aSARS-CoV-2 encoded polypeptide as described in WO 2021/159040,including, e.g., an N-terminal domain (NTD) of a SARS-CoV-2 Spikeprotein, a receptor binding domain (RBD) of a SARS-CoV-2 Spike protein,Heptapeptide repeat sequence 1 (HR1) of a SARS-CoV-2 Spike protein,Heptapeptide repeat sequence 2 (HR1) of a SARS-CoV-2 Spike protein,and/or combinations thereof). Among other things, the present disclosureparticularly documents surprising and useful characteristics and/oradvantages of certain RNA constructs (e.g., in some embodiments mRNAconstructs) encoding a SARS-CoV-2 RBD portion and, in some embodiments,not encoding a full length SARS-CoV-2 S protein. Without wishing to bebound by any particular theory, the present disclosure suggests thatprovided RNA constructs (e.g., in some embodiments mRNA constructs) thatencode less than a full-length SARS-CoV-2 S protein, and particularlythose that encode at least an RBD portion of such SARS-CoV-2 S proteinmay be particularly useful and/or effective for use as or in animmunogenic composition (e.g., a vaccine), and/or for achievingimmunological effects as described herein (e.g., generation ofSARS-CoV-2 neutralizing antibodies, and/or T cell responses (e.g., CD4+and/or CD8+ T cell responses)).

In some embodiments, the present disclosure provides an RNA (e.g., mRNA)comprising an open reading frame encoding a polypeptide that comprises areceptor-binding portion of a SARS-CoV-2 S protein, which RNA issuitable for intracellular expression of the polypeptide. In someembodiments, such an encoded polypeptide does not comprise the completeS protein. In some embodiments, an encoded polypeptide comprises thereceptor binding domain (RBD), for example, as shown in SEQ ID NO: 5. Insome embodiments, the encoded polypeptide comprises the peptideaccording to SEQ ID NO: 29 or 31. In some embodiments, such an RNA(e.g., mRNA) may be complexed by a (poly)cationic polymer, polyplex(es),protein(s) or peptide(s). In some embodiments, such an RNA may beformulated in a lipid nanoparticle (e.g., ones described herein). Insome embodiments, such an RNA (e.g., mRNA) may be particularly usefuland/or effective for use as or in an immunogenic composition (e.g., avaccine), and/or for achieving immunological effects as described herein(e.g., generation of SARS-CoV-2 neutralizing antibodies, and/or T cellresponses (e.g., CD4+ and/or CD8+ T cell responses)). In someembodiments, such an RNA (e.g., mRNA) may be useful for vaccinatinghumans (including, e.g., humans known to have been exposed and/orinfected by SARS-CoV-2, and/or humans not known to have been exposed toSARS-CoV-2).

Those skilled in the art, reading the present disclosure, will furtherappreciate that it describes various mRNA constructs comprising anucleic acid sequence that encodes a full-length SARS-CoV-2 Spikeprotein (e.g., including embodiments in which such encoded SARS-CoV-2Spike protein may comprise at least one or more amino acidsubstitutions, e.g., proline substitutions as described herein, and/orembodiments in which the mRNA sequence is codon-optimized e.g., formammalian, e.g., human, subjects). In some embodiments, such afull-length SARS-CoV-2 Spike protein may have an amino acid sequencethat is or comprises that set forth in SEQ ID NO: 7. Still further, sucha person of ordinary skill, reading the present disclosure, willappreciate, among other things, that it describes particularcharacteristics and/or advantages of certain mRNA constructs comprisinga nucleic acid sequence that encodes a full-length SARS-CoV-2 Spikeprotein. Without wishing to be bound by any particular theory, thepresent disclosure suggests that provided mRNA constructs that encode afull-length SARS-CoV-2 S protein may be particularly useful and/oreffective for use as or in an immunogenic composition (e.g., a vaccine)in particular subject population (e.g., particular age populations). Forexample, in some embodiments, such an mRNA composition may beparticularly useful in younger (e.g., less than 25 years old, 20 yearsold, 18 years old, 15 years, 10 years old, or lower) subjects;alternatively or additionally, in some embodiments, such an mRNAcomposition may be particularly useful in elderly subjects (e.g., over55 years old, 60 years old, 65 years old, 70 years old, 75 years old, 80years old, 85 years old, or higher). In particular embodiments, animmunogenic composition comprising such an mRNA construct providedherein exhibits a minimal to modest increase (e.g., no more than 30%increase, no more than 20% increase, or no more than 10% increase, orlower) in dose level and/or dose number-dependent systemicreactogenicity (e.g., fever, fatigue, headache, chills, diarrhea, musclepain, and/or joint pain, etc.) and/or local tolerability (e.g., pain,redness, and/or swelling, etc.), at least in some subjects (e.g., insome subject age groups); in some embodiments, such reactogenicityand/or local tolerability is observed particularly, in in younger agegroup (e.g., less than 25 years old, 20 years old, 18 years old orlower) subjects, and/or in older (e.g., elderly) age group (e.g., 65-85years old). In some embodiments, provided mRNA constructs that encode afull-length SARS-CoV-2 S protein may be particularly useful and/oreffective for use as or in an immunogenic composition (e.g., a vaccine)for inducing SARS-CoV-2 neutralizing antibody response level in apopulation of subjects that are at high risk for severe diseasesassociated with SARS-CoV-2 infection (e.g., an elderly population, forexample, 65-85 year-old group). In some embodiments, a person ofordinary skill, reading the present disclosure, will appreciate, amongother things, that provided mRNA constructs that encode a full-lengthSARS-CoV-2 S protein, which exhibit a favorable reactogenicity profile(e.g., as described herein) in younger and elderly age populations, maybe particularly useful and/or effective for use as or in an immunogeniccomposition (e.g., a vaccine) for achieving immunological effects asdescribed herein (e.g., generation of SARS-CoV-2 neutralizingantibodies, and/or T cell responses (e.g., CD4+ and/or CD8+ T cellresponses)). In some embodiments, the present disclosure also suggeststhat provided mRNA constructs that encode a full-length SARS-CoV-2 Sprotein may be particularly effective to protect against SARS-CoV-2infection, as characterized by earlier clearance of SARS-CoV-2 viral RNAin non-human mammalian subjects (e.g., rhesus macaques) that wereimmunized with immunogenic compositions comprising such mRNA constructsand subsequently challenged by SARS-CoV-2 strain. In some embodiments,such earlier clearance of SARS-CoV-2 viral RNA may be observed in thenose of non-human mammalian subjects (e.g., rhesus macaques) that wereimmunized with immunogenic compositions comprising such mRNA constructsand subsequently challenged by SARS-CoV-2 strain.

In some embodiments, the present disclosure provides an RNA (e.g., mRNA)comprising an open reading frame encoding a full-length SARS-CoV-2 Sprotein (e.g., a full-length SARS-CoV-2 S protein with one or more aminoacid substitutions), which RNA is suitable for intracellular expressionof the polypeptide. In some embodiments, the encoded polypeptidecomprises the amino acid sequence of SEQ ID NO:7. In some embodiments,such an RNA (e.g., mRNA) may be complexed by a (poly)cationic polymer,polyplex(es), protein(s) or peptide(s). In some embodiments, such an RNAmay be formulated in a lipid nanoparticle (e.g., ones described herein).

In some embodiments, an immunogenic composition provided herein maycomprise a plurality of (e.g., at least two or more, including, e.g., atleast three, at least four, at least five, at least six, at least seven,at least eight, at least nine, at least ten, etc.) immunoreactiveepitopes of a SARS-CoV-2 polypeptide or variants thereof. In some suchembodiments, such a plurality of immunoreactive epitopes may be encodedby a plurality of RNAs (e.g., mRNAs). In some such embodiments, such aplurality of immunoreactive epitopes may be encoded by a single RNA(e.g., mRNA). In some embodiments, nucleic acid sequences encoding aplurality of immunoreactive epitopes may be separated from each other ina single RNA (e.g., mRNA) by a linker (e.g., a peptide linker in someembodiments). Without wishing to be bound by any particular theory, insome embodiments, provided polyepitope immunogenic compositions(including, e.g., those that encode a full-length SARS-CoV-2 spikeprotein) may be particularly useful, when considering the geneticdiversity of SARS-CoV-2 variants, to provide protection against numerousviral variants and/or may offer a greater opportunity for development ofa diverse and/or otherwise robust (e.g., persistent, e.g., detectableabout 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more days afteradministration of one or more doses) neutralizing antibody and/or T cellresponse, and in particular a particularly robust T_(H)1-type T cell(e.g., CD4+ and/or CD8+ T cell) response.

In some embodiments, the present disclosure documents that providedcompositions and/or methods are characterized by (e.g., whenadministered to a relevant population, which may in some embodiments beor comprise an adult population) in that they achieve one or moreparticular therapeutic outcomes (e.g., effective immune responses asdescribed herein and/or detectable expression of encoded SARS-CoV-2 Sprotein or an immunogenic fragment thereof) with a singleadministration; in some such embodiments, an outcome may be assessed,for example, as compared to that observed in absence of RNA vaccines(e.g., mRNA vaccines) described herein. In some embodiments, aparticular outcome may be achieved at a lower dose than required for oneor more alternative strategies.

In some embodiments, the present disclosure provides an immunogeniccomposition comprising an isolated messenger ribonucleic acid (mRNA)polynucleotide, wherein the isolated mRNA polynucleotide comprises anopen reading frame encoding a polypeptide that comprises areceptor-binding portion of a SARs-CoV-2 S protein, and wherein theisolated mRNA polynucleotide is formulated in at least one lipidnanoparticle. For example, in some embodiments, such a lipidnanoparticle may comprise a molar ratio of 20-60% ionizable cationiclipid, 5-25% non-cationic lipid (e.g., neutral lipid), 25-55% sterol orsteroid, and 0.5-15% polymer-conjugated lipid (e.g., PEG-modifiedlipid). In some embodiments, a sterol or steroid included in a lipidnanoparticle may be or comprise cholesterol. In some embodiments, aneutral lipid may be or comprise1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In some embodiments,a polymer-conjugated lipid may be or comprise PEG2000 DMG. In someembodiments, such an immunogenic composition may comprise a total lipidcontent of about 1 mg to 10 mg, or 3 mg to 8 mg, or 4 mg to 6 mg. Insome embodiments, such an immunogenic composition may comprise a totallipid content of about 5 mg/mL-15 mg/mL or 7.5 mg/mL-12.5 mg/mL or 9-11mg/mL. In some embodiments, such an isolated mRNA polynucleotide isprovided in an effective amount to induce an immune response in asubject administered at least one dose of the immunogenic composition.In some embodiments, a polypeptide encoded by a provided isolated mRNApolynucleotide does not comprise the complete S protein. In someembodiments, such an isolated mRNA polynucleotide provided in animmunogenic composition is not self-replicating RNA.

In some embodiments, an immune response may comprise generation of abinding antibody titer against SARS-CoV-2 protein (including, e.g., astabilized prefusion spike trimer in some embodiments) or a fragmentthereof. In some embodiments, an immune response may comprise generationof a binding antibody titer against the receptor binding domain (RBD) ofthe SARS-CoV-2 spike protein. In some embodiments, a providedimmunogenic composition has been established to achieve a detectablebinding antibody titer after administration of a first dose, withseroconversion in at least 70% (including, e.g., at least 80%, at least90%, at least 95% and up to 100%) of a population of subjects receivingsuch a provided immunogenic composition, for example, by about 2 weeks.

In some embodiments, an immune response may comprise generation of aneutralizing antibody titer against SARS-CoV-2 protein (including, e.g.,a stabilized prefusion spike trimer in some embodiments) or a fragmentthereof. In some embodiments, an immune response may comprise generationof a neutralizing antibody titer against the receptor binding domain(RBD) of the SARS-CoV-2 spike protein. In some embodiments, a providedimmunogenic composition has been established to achieve a neutralizingantibody titer in an appropriate system (e.g., in a human infected withSARS-CoV-2 and/or a population thereof, and/or in a model systemtherefor). For example, in some embodiments, such neutralizing antibodytiter may have been demonstrated in one or more of a population ofhumans, a non-human primate model (e.g., rhesus macaques), and/or amouse model.

In some embodiments, a neutralizing antibody titer is a titer that is(e.g., that has been established to be) sufficient to reduce viralinfection of B cells relative to that observed for an appropriatecontrol (e.g., an unvaccinated control subject, or a subject vaccinatedwith a live attenuated viral vaccine, an inactivated viral vaccine, or aprotein subunit viral vaccine, or a combination thereof). In some suchembodiments, such reduction is of at least 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more.

In some embodiments, a neutralizing antibody titer is a titer that is(e.g., that has been established to be) sufficient to reduce the rate ofasymptomatic viral infection relative to that observed for anappropriate control (e.g., an unvaccinated control subject, or a subjectvaccinated with a live attenuated viral vaccine, an inactivated viralvaccine, or a protein subunit viral vaccine, or a combination thereof).In some such embodiments, such reduction is of at least 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. In someembodiments, such reduction can be characterized by assessment ofSARS-CoV-2 N protein serology. Significant protection againstasymptomatic infection was also confirmed by real life observations (seealso: Dagan N. et al., N Engl J Med. 2021, doi: 10.1056/NEJMoa2101765.Epub ahead of print. PMID: 33626250)

In some embodiments, a neutralizing antibody titer is a titer that is(e.g., that has been established to be) sufficient to reduce or blockfusion of virus with epithelial cells and/or B cells of a vaccinatedsubject relative to that observed for an appropriate control (e.g., anunvaccinated control subject, or a subject vaccinated with a liveattenuated viral vaccine, an inactivated viral vaccine, or a proteinsubunit viral vaccine, or a combination thereof). In some suchembodiments, such reduction is of at least 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more.

In some embodiments, induction of a neutralizing antibody titer may becharacterized by an elevation in the number of B cells, which in someembodiments may include plasma cells, class-switched IgG1- andIgG2-positive B cells, and/or germinal center B cells. In someembodiments, a provided immunogenic composition has been established toachieve such an elevation in the number of B cells in an appropriatesystem (e.g., in a human infected with SARS-CoV-2 and/or a populationthereof, and/or in a model system therefor). For example, in someembodiments, such an elevation in the number of B cells may have beendemonstrated in one or more of a population of humans, a non-humanprimate model (e.g., rhesus macaques), and/or a mouse model. In someembodiments, such an elevation in the number of B cells may have beendemonstrated in draining lymph nodes and/or spleen of a mouse modelafter (e.g., at least 7 days, at least 8 days, at least 9 days, at least10 days, at least 11 days, at least 12 days, at least 13 days, at least14 days, after) immunization of such a mouse model with a providedimmunogenic composition.

In some embodiments, induction of a neutralizing antibody titer may becharacterized by a reduction in the number of circulating B cells inblood. In some embodiments, a provided immunogenic composition has beenestablished to achieve such a reduction in the number of circulating Bcells in blood of an appropriate system (e.g., in a human infected withSARS-CoV-2 and/or a population thereof, and/or in a model systemtherefor). For example, in some embodiments, such a reduction in thenumber of circulating B cells in blood may have been demonstrated in oneor more of a population of humans, a non-human primate model (e.g.,rhesus macaques), and/or a mouse model. In some embodiments, such areduction in the number of circulating B cells in blood may have beendemonstrated in a mouse model after (e.g., at least 4 days, at least 5days, at least 6 days, at least 7 days, at least 8 days, at least 9days, at least 10 days, after) immunization of such a mouse model with aprovided immunogenic composition. Without wishing to be bound by theory,a reduction in circulating B cells in blood may be due to B cell homingto lymphoid compartments.

In some embodiments, an immune response induced by a providedimmunogenic composition may comprise an elevation in the number of Tcells. In some embodiments, such an elevation in the number of T cellsmay include an elevation in the number of T follicular helper (T_(FH))cells, which in some embodiments may comprise one or more subsets withICOS upregulation. One of skilled in the art will understand thatproliferation of T_(FH) in germinal centres is integral for generationof an adaptive B-cell response, and also that in humans, T_(FH)occurring in the circulation after vaccination is typically correlatedwith a high frequency of antigen-specific antibodies. In someembodiments, a provided immunogenic composition has been established toachieve such an elevation in the number of T cells (e.g., T_(FH) cells)in an appropriate system (e.g., in a human infected with SARS-CoV-2and/or a population thereof, and/or in a model system therefor). Forexample, in some embodiments, such an elevation in the number of T cells(e.g., T_(FH) cells) may have been demonstrated in one or more of apopulation of humans, a non-human primate model (e.g., rhesus macaques),and/or a mouse model. In some embodiments, such an elevation in thenumber of T cells (e.g., e.g., T_(FH) cells) may have been demonstratedin draining lymph nodes, spleen, and/or blood of a mouse model after(e.g., at least 4 days, at least 5 days, at least 6 days, at least 7days, at least 8 days, at least 9 days, at least 10 days, at least 11days, at least 12 days, at least 13 days, at least 14 days, after)immunization of such a mouse model with a provided immunogeniccomposition. In some embodiments, a protective response againstSARS-CoV-2 induced by a provided immunogenic composition has beenestablished in an appropriate model system for SARS-CoV-2. For example,in some embodiments, such a protective response may have beendemonstrated in an animal model, e.g., a non-human primate model (e.g.,rhesus macaques) and/or a mouse model. In some embodiments, a non-humanprimate (e.g., rhesus macaque) or a population thereof that has/havereceived at least one immunization with a provided immunogeniccomposition is/are challenged with SARS-CoV-2, e.g., through intranasaland/or intratracheal route. In some embodiments, such a challenge may beperformed several weeks (e.g., 5-10 weeks) after at least oneimmunization (including, e.g., at least two immunizations) with aprovided immunogenic composition. In some embodiments, such a challengemay be performed when a detectable level of a SARS-CoV-2 neutralizingtiter (e.g., antibody response to SARS-CoV-2 spike protein and/or afragment thereof, including, e.g., but not limited to a stabilizedprefusion spike trimer, S-2P, and/or antibody response toreceptor-binding portion of SARS-CoV-2) is achieved in non-humanprimate(s) (e.g., rhesus macaque(s)) that has received at least oneimmunization (including, e.g., at least two immunizations) with aprovided immunogenic composition. In some embodiments, a protectiveresponse is characterized by absence of or reduction in detectable viralRNA in bronchoalveolar lavage (BAL) and/or nasal swabs of challengednon-human primate(s) (e.g., rhesus macaque(s)). In some embodiments,immunogenic compositions described herein may have been characterized inthat a larger percent of challenged animals, for example, non-humanprimates in a population (e.g., rhesus macaques), that have received atleast one immunization (including, e.g., at least two immunizations)with a provided immunogenic composition display absence of detectableRNA in their BAL and/or nasal swab, as compared to a population ofnon-immunized animals, for example, non-human primates (e.g., rhesusmacaques). In some embodiments, immunogenic compositions describedherein may have been characterized in that challenged animals, forexample, non-human in a population (e.g., rhesus macaques), that havereceived at least one immunization (including, e.g., at least twoimmunizations) with a provided immunogenic composition may showclearance of viral RNA in nasal swab no later than 10 days, including,e.g., no later than 8 days, no later than 6 days, no later than 4 days,etc., as compared to a population of non-immunized animals, for example,non-human primates (e.g., rhesus macaques).

In some embodiments, immunogenic compositions described herein whenadministered to subjects in need thereof do not substantially increasethe risk of vaccine-associated enhanced respiratory disease. In someembodiments, such vaccine-associated enhanced respiratory disease may beassociated with antibody-dependent enhancement of replication and/orwith vaccine antigens that induced antibodies with poor neutralizingactivity and Th2-biased responses. In some embodiments, immunogeniccompositions described herein when administered to subjects in needthereof do not substantially increase the risk of antibody-dependentenhancement of replication.

In some embodiments, a single dose of an RNA composition (e.g., mRNAformulated in lipid nanoparticles) can induce a therapeutic antibodyresponse in less than 10 days of vaccination. In some embodiments, sucha therapeutic antibody response may be characterized in that when suchan RNA vaccine can induce production of about 10-100 ug/mL IgG measuredat 10 days after vaccination at a dose of 0.1 to 10 ug or 0.2-5 ug in ananimal model. In some embodiments, such a therapeutic antibody responsemay be characterized in that such an RNA vaccine induces about 100-1000ug/mL IgG measured at 20 days of vaccination at a dose of 0.1 to 10 ugor 0.2-5 ug in an animal model. In some embodiments, a single dose mayinduce a pseudovirus-neutralization titer, as measured in an animalmodel, of 10-200 pVN50 titer 15 days after vaccination. In someembodiments, a single dose may induce a pseudovirus-neutralizationtiter, as measured in an animal model, of 50-500 pVN50 titer 15 daysafter vaccination.

In some embodiments, a single dose of an RNA composition (e.g., mRNAcomposition) can expand antigen-specific CD8 and/or CD4 T cell responseby at least at 50% or more (including, e.g., at least 60%, at least 70%,at least 80%, at least 90%, at least 95%, or more), as compared to thatobserved in absence of such an RNA construct encoding a SARS-COV2immunogenic protein or fragment thereof (e.g., spike protein and/orreceptor binding domain). In some embodiments, a single dose of an RNAcomposition can expand antigen-specific CD8 and/or CD4 T cell responseby at least at 1.5-fold or more (including, e.g., at least 2-fold, atleast 3-fold, at least 5-fold, at least 10-fold, at least 50-fold, atleast 100-fold, at least 500-fold, at least 1000-fold, or more), ascompared to that observed in absence of such an RNA construct encoding aSARS-COV2 immunogenic protein or fragment thereof (e.g., spike proteinand/or receptor binding domain).

In some embodiments, a regimen (e.g., a single dose of an mRNAcomposition) can expand T cells that exhibit a Th1 phenotype (e.g., ascharacterized by expression of IFN-gamma, IL-2, IL-4, and/or IL-5) by atleast at 50% or more (including, e.g., at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, or more), as compared to thatobserved in absence of such an mRNA construct encoding a SARS-COV2immunogenic protein or fragment thereof (e.g., spike protein and/orreceptor binding domain). In some embodiments, a regimen (e.g., a singledose of an mRNA composition) can expand T cells that exhibit a Th1phenotype (e.g., as characterized by expression of IFN-gamma, IL-2,IL-4, and/or IL-5), for example by at least at 1.5-fold or more(including, e.g., at least 2-fold, at least 3-fold, at least 5-fold, atleast 10-fold, at least 50-fold, at least 100-fold, at least 500-fold,at least 1000-fold, or more), as compared to that observed in absence ofsuch an mRNA construct encoding a SARS-COV2 immunogenic protein orfragment thereof (e.g., spike protein and/or receptor binding domain).In some embodiments, a T-cell phenotype may be or comprise aTh1-dominant cytokine profile (e.g., as characterized by INF-gammapositive and/or IL-2 positive), and/or no by or biologicallyinsignificant IL-4 secretion.

In some embodiments, a regimen as described herein (e.g., one or moredoses of an mRNA composition) induces and/or achieves production ofRBD-specific CD4+ T cells. Among other things, the present disclosuredocuments that mRNA compositions encoding an RBD-containing portion of aSARS-CoV-2 spike protein (e.g., and not encoding a full-lengthSARS-CoV-2 spike protein) may be particularly useful and/or effective insuch induction and/or production of RBD-specific CD4+ T cells. In someembodiments, RBD-specific CD4+ T-cells induced by an mRNA compositiondescribed herein (e.g., by an mRNA composition that encodings anRBD-containing-portion of a SARS-CoV-2 spike protein and, in someembodiments not encoding a full-length SARS-CoV-2 spike protein)demonstrate a Th1-dominant cytokine profile (e.g., as characterized byINF-gamma positive and/or IL-2 positive), and/or by no or biologicallyinsignificant IL-4 secretion.

In some embodiments, characterization of CD4+ and/or CD8+ T cellresponses (e.g., described herein) in subjects receiving RNAcompositions (e.g., as described herein) may be performed using ex vivoassays using PBMCs collected from the subjects.

In some embodiments, immunogenicity of RNA (e.g., mRNA) compositionsdescribed herein may be assessed by one of or more of the followingserological immunongenicity assays: detection of IgG, IgM, and/or IgA toSARS-CoV-2 S protein present in blood samples of a subject receiving aprovided RNA composition, and/or neutralization assays using SARS-CoV-2pseudovirus and/or a wild-type SARS-CoV-2 virus.

In some embodiments, an RNA composition (e.g., as described herein)provide a relatively low adverse effect (e.g., Grade 1-Grade 2 pain,redness and/or swelling) within 7 days after vaccinations at a dose of10 ug-100 ug or 1 ug-50 ug. In some embodiments, RNA compositions (e.g.,as described herein) provide a relatively low observation of systemicevents (e.g., Grade 1-Grade 2 fever, fatigue, headache, chills,vomiting, diarrhea, muscle pain, joint pain, medication, andcombinations thereof) within 7 days after vaccinations at a dose of 10ug-100 ug.

In some embodiments, RNA (e.g., mRNA) compositions are characterized inthat when administered to subjects at 10-100 ug dose or 1 ug-50 ug, IgGdirected to a SARS-CoV2 immunogenic protein or fragment thereof (e.g.,spike protein and/or receptor binding domain) may be produced at a levelof 100-100,000 U/mL or 500-50,000 U/mL 21 days after vaccination.

In some embodiments, an RNA (e.g., mRNA) encodes a natively-foldedtrimeric receptor binding protein of SARS-CoV-2. In some embodiments, anRNA (e.g., mRNA) encodes a variant of such receptor binding protein suchthat the encoded variant binds to ACE2 at a Kd of 10 pM or lower,including, e.g., at a Kd of 9 pM, 8 pM, 7 pM, 6 pM, 5 pM, 4 pM, orlower. In some embodiments, an RNA (e.g., mRNA) encodes a variant ofsuch receptor binding protein such that the encoded variant binds toACE2 at a Kd of 5 pM. In some embodiments, an RNA (e.g., mRNA) encodes atrimeric receptor binding portion of SARS-CoV-2 that comprises an ACE2receptor binding site. In some embodiments, an RNA (e.g., mRNA)comprises a coding sequence for a receptor-binding portion of SARS-CoV-2and a trimerization domain (e.g., a natural trimerization domain(foldon) of T4 fibritin) such that the coding sequence directsexpression of a trimeric protein that has an ACE2 receptor binding siteand binds ACE2. In some embodiments, an RNA (e.g., mRNA) encodes atrimeric receptor binding portion of SARS-CoV-2 or a variant thereofsuch that its Kd is smaller than that for a monomeric receptor-bindingdomain (RBD) of SARS-CoV-2. For example, in some embodiments, an RNA(e.g., mRNA) encodes a trimeric receptor binding portion of SARS-CoV-2or a variant thereof such that its Kd is at least 10-fold (including,e.g., at least 50-fold, at least 100-fold, at least 500-fold, at least1000-fold, etc.) smaller than that for a RBD of SARS-CoV-2.

In some embodiments, a trimer receptor binding portion of SARS-CoV-2encoded by an RNA (e.g., as described herein) may be determined to havea size of about 3-4 angstroms when it is complexed with ACE2 and B⁰AT1neutral amino acid transporter in a closed conformation, ascharacterized by electron cryomicroscopy (cryoEM). In some embodiments,geometric mean SARS-CoV-2 neutralizing titer that characterizes and/oris achieved by an RNA composition or method as described herein canreach at least 1.5-fold, including, at least 2-fold, at least 2.5-fold,at least 3-fold, or higher, that of a COVID-19 convalescent human panel(e.g., a panel of sera from COVID-19 convalescing humans obtained 20-40days after the onset of symptoms and at least 14 days after the start ofasymptomatic convalescence.

In some embodiments, RNA compositions as provided herein may becharacterized in that subjects who have been treated with suchcompositions (e.g., with at least one dose, at least two doses, etc) mayshow reduced and/or more transient presence of viral RNA in relevantsite(s) (e.g., nose and/or lungs, etc, and/or any other tissuesusceptible to infection) as compared with an appropriate control (e.g.,an established expected level for a comparable subject or population nothaving been so treated and having been exposed to virus under reasonablycomparable exposure conditions)

In some embodiments, the RBD antigen expressed by an mRNA construct(e.g., as described herein) can be modified by addition of aT4-fibritin-derived “foldon” trimerization domain, for example, toincrease its immunogenicity.

In some embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that certain local reactions(e.g., pain, redness, and/or swelling, etc.) and/or systemic events(e.g., fever, fatigue, headache, etc.) may appear and/or peak at Day 2after vaccination. In some embodiments, RNA (e.g., mRNA) compositionsdescribed herein are characterized in that certain local reactions(e.g., pain, redness, and/or swelling, etc.) and/or systemic events(e.g., fever, fatigue, headache, etc.) may resolve by Day 7 aftervaccination.

In some embodiments, RNA compositions (e.g., mRNA) and/or methodsdescribed herein are characterized in that no Grade 1 or greater changein routine clinical laboratory values or laboratory abnormalities areobserved in subjects receiving RNA compositions (e.g., as describedherein). Examples of such clinical laboratory assays may includelymphocyte count, hematological changes, etc.

In some embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that by 21 days after a first dose(e.g., 10-100 ug inclusive or 1 ug-50 ug inclusive), geometric meanconcentrations (GMCs) of IgG directed to a SARS-CoV-2 S polypeptide oran immunogenic fragment thereof (e.g., RBD) may reach 200-3000 units/mLor 500-3000 units/mL or 500-2000 units/mL, compared to 602 units/mL fora panel of COVID-19 convalescent human sera. In some embodiments, RNA(e.g., mRNA) compositions described herein are characterized in that by7 days after a second dose (e.g., 10-30 ug inclusive; or 1 ug-50 uginclusive), geometric mean concentrations (GMCs) of IgG directed to aSARS-CoV-2 spike polypeptide or an immunogenic fragment thereof (e.g.,RBD) may increase by at least 8-fold or higher, including, e.g., atleast 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, atleast 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, orhigher. In some embodiments, RNA (e.g., mRNA) compositions describedherein are characterized in that by 7 days after a second dose (e.g.,10-30 ug inclusive; or 1 ug-50 ug inclusive), geometric meanconcentrations (GMCs) of IgG directed to a SARS-CoV-2 S polypeptide oran immunogenic fragment thereof (e.g., RBD) may increase to 1500units/mL to 40,000 units/mL or 4000 units/mL to 40,000 units/mL. In someembodiments, antibody concentrations described herein can persist to atleast 20 days or longer, including, e.g., at least 25 days, at least 30days, at least 35 days, at least 40 days, at least 45 days, at least 50days, after a first dose, or at least 10 days or longer, including,e.g., at least 15 days, at least 20 days, at least 25 days, or longer,after a second dose. In some embodiments, antibody concentrations canpersist to 35 days after a first dose, or at least 14 days after asecond dose.

In some embodiments, RNA (e.g., mRNA) compositions described herein arecharacterized in that when measured at 7 days after a second dose (e.g.,1-50 ug inclusive), GMC of IgG directed to a SARS-CoV-2 S polypeptide oran immunogenic fragment thereof (e.g., RBD) is at least 30% higher(including, e.g., at least 40% higher, at least 50% higher, at least60%, higher, at least 70% higher, at least 80% higher, at least 90%higher, at least 95% higher, as compared to antibody concentrationsobserved in a panel of COVID-19 convalescent human serum. In manyembodiments, geometric mean concentration (GMC) of IgG described hereinis GMCs of RBD-binding IgG.

In some embodiments, RNA (e.g., mRNA) compositions described herein arecharacterized in that when measured at 7 days after a second dose (e.g.,10-50 ug inclusive), GMC of IgG directed to a SARS-CoV-2 S polypeptideor an immunogenic fragment thereof (e.g., RBD) is at least 1.1-foldhigher (including, e.g., at least 1.5-fold, at least 2-fold, at least3-fold, at least 4-fold, at least 5-fold, at least 6-fold higher, atleast 7-fold higher, at least 8-fold higher, at least 9-fold higher, atleast 10-fold higher, at least 15-fold higher, at least 20-fold higher,at least 25-fold higher, at least 30-fold higher), as compared toantibody concentrations observed in a panel of COVID-19 convalescenthuman serum, In many embodiments, geometric mean concentration (GMC) ofIgG described herein is GMCs of RBD-binding IgG.

In some embodiments, RNA (e.g., mRNA) compositions described herein arecharacterized in that when measured at 21 days after a second dose, GMCof IgG directed to a SARS-CoV-2 S polypeptide or an immunogenic fragmentthereof (e.g., RBD) is at least 5-fold higher (including, e.g., at least6-fold higher, at least 7-fold higher, at least 8-fold higher, at least9-fold higher, at least 10-fold higher, at least 15-fold higher, atleast 20-fold higher, at least 25-fold higher, at least 30-fold higher),as compared to antibody concentrations observed in a panel of COVID-19convalescent human serum, In many embodiments, geometric meanconcentration (GMC) of IgG described herein is GMCs of RBD-binding IgG.

In some embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that an increase (e.g., at least30%, at least 40%, at least 50%, or more) in SARS-CoV-2 neutralizinggeometric mean titers (GMTs) is observed 21 days after a first dose. Insome embodiments, RNA (e.g., mRNA) compositions described herein arecharacterized in that a substantially greater serum neutralizing GMTsare achieved 7 days after subjects receive a second dose (e.g., 10 μg-30μg inclusive), reaching 150-300, compared to 94 for a COVID-19convalescent serum panel.

In some embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that 7 days after administrationof the second dose, the protective efficacy is at least 60%, e.g., atleast 70%, at least 80%, at least 90, or at least 95%. In oneembodiment, RNA (e.g., mRNA) compositions and/or methods describedherein are characterized in that 7 days after administration of thesecond dose, the protective efficacy is at least 70%. In one embodiment,RNA (e.g., mRNA) compositions and/or methods described herein arecharacterized in that 7 days after administration of the second dose,the protective efficacy is at least 80%. In one embodiment, RNA (e.g.,mRNA) compositions and/or methods described herein are characterized inthat 7 days after administration of the second dose, the protectiveefficacy is at least 90%. In one embodiment, RNA (e.g., mRNA)compositions and/or methods described herein are characterized in that 7days after administration of the second dose, the protective efficacy isat least 95%.

In some embodiments, an RNA composition provided herein is characterizedin that it induces an immune response against SARS-CoV-2 after at least7 days after a dose (e.g., after a second dose). In some embodiments, anRNA composition provided herein is characterized in that it induces animmune response against SARS-CoV-2 in less than 14 days after a dose(e.g., after a second dose). In some embodiments, an RNA compositionprovided herein is characterized in that it induces an immune responseagainst SARS-CoV-2 after at least 7 days after a vaccination regimen. Insome embodiments, a vaccination regimen comprises a first dose and asecond dose. In some embodiments, a first dose and a second dose areadministered by at least 21 days apart. In some such embodiments, animmune response against SARS-CoV-2 is induced at least after 28 daysafter a first dose.

In some embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that geometric mean concentration(GMCs) of antibodies directed to a SARS-CoV-2 spike polypeptide or animmunogenic fragment thereof (e.g., RBD), as measured in serum fromsubjects receiving RNA (e.g., mRNA) compositions of the presentdisclosure (e.g., at a dose of 10-30 ug inclusive), is substantiallyhigher than in a convalescent serum panel (e.g., as described herein).In some embodiments where a subject may receive a second dose (e.g., 21days after 1 first dose), geometric mean concentration (GMCs) ofantibodies directed to a SARS-CoV-2 spike polypeptide or an immunogenicfragment thereof (e.g., RBD), as measured in serum from the subject, maybe 8.0-fold to 50-fold higher than a convalescent serum panel GMC. Insome embodiments where a subject may receive a second dose (e.g., 21days after 1 first dose), geometric mean concentration (GMCs) ofantibodies directed to a SARS-CoV-2 spike polypeptide or an immunogenicfragment thereof (e.g., RBD), as measured in serum from the subject, maybe at least 8.0-fold or higher, including, e.g., at least 10-fold, atleast 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, atleast 60-fold or higher, as compared to a convalescent serum panel GMC.

In some embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that the SARS-CoV-2 neutralizinggeometric mean titer, as measured at 28 days after a first dose or 7days after a second dose, may be at least 1.5-fold or higher (including,e.g., at least 2-fold, at least 2.5-fold, at least 3-fold, at least3.5-fold or higher), as compared to a neutralizing GMT of a convalescentserum panel.

In some embodiments, a regimen administered to a subject may be orcomprise a single dose. In some embodiments, a regimen administered to asubject may comprise a plurality of doses (e.g., at least two doses, atleast three doses, or more). In some embodiments, a regimen administeredto a subject may comprise a first dose and a second dose, which aregiven at least 2 weeks apart, at least 3 weeks apart, at least 4 weeksapart, or more. In some embodiments, such doses may be at least 1 month,at least 2 months, at least 3 months, at least 4 months, at least 5months, at least 6 months, at least 7 months, at least 8 months, atleast 9 months, at least 10 months, at least 11 months, at least 12months, or more apart. In some embodiments, doses may be administereddays apart, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60 or more days apart. In someembodiments, doses may be administered about 1 to about 3 weeks apart,or about 1 to about 4 weeks apart, or about 1 to about 5 weeks apart, orabout 1 to about 6 weeks apart, or about 1 to more than 6 weeks apart.In some embodiments, doses may be separated by a period of about 7 toabout 60 days, such as for example about 14 to about 48 days, etc. Insome embodiments, a minimum number of days between doses may be about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21or more. In some embodiments, a maximum number of days between doses maybe about 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45,44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27,26, 25, 24, 23, 22, 21, or fewer. In some embodiments, doses may beabout 21 to about 28 days apart. In some embodiments, doses may be about19 to about 42 days apart. In some embodiments, doses may be about 7 toabout 28 days apart. In some embodiments, doses may be about 14 to about24 days. In some embodiments, doses may be about 21 to about 42 days.

In some embodiments, particularly for compositions established toachieve elevated antibody and/or T-cell titres for a period of timelonger than about 3 weeks—e.g., in some embodiments, a providedcomposition is established to achieve elevated antibody and/or T-celltitres (e.g., specific for a relevant portion of a SARS-CoV-2 spikeprotein) for a period of time longer than about 3 weeks; in some suchembodiments, a dosing regimen may involve only a single dose, or mayinvolve two or more doses, which may, in some embodiments, be separatedfrom one another by a period of time that is longer than about 21 daysor three weeks. For example, in some such embodiments, such period oftime may be about 4 weeks, weeks, 6 weeks 7 weeks, 8 weeks, 9 weeks, 10weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 wees, 16 weeks, 17weeks, 18 weeks, 19 weeks, 20 weeks or more, or about 1 month, 2 months,3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months,10, months, 11 months, 12 months or more, or in some embodiments about ayear or more. In some embodiments, a first dose and a second dose(and/or other subsequent dose) may be administered by intramuscularinjection. In some embodiments, a first dose and a second dose may beadministered in the deltoid muscle. In some embodiments, a first doseand a second dose may be administered in the same arm. In someembodiments, an RNA (e.g., mRNA) composition described herein isadministered (e.g., by intramuscular injection) as a series of two doses(e.g., 0.3 mL each) 21 days part. In some embodiments, each dose isabout 30 ug. In some embodiments, each dose may be higher than 30 ug,e.g., about 40 ug, about 50 ug, about 60 ug. In some embodiments, eachdose may be lower than 30 ug, e.g., about 20 ug, about 10 ug, about 5ug, etc. In some embodiments, each dose is about 3 ug or lower, e.g.,about 1 ug. In some such embodiments, an RNA (e.g., mRNA) compositiondescribed herein is administered to subjects of age 16 or older(including, e.g., 16-85 years). In some such embodiments, an RNAcomposition (e.g., mRNA) described herein is administered to subjects ofage 18-55. In some such embodiments, an RNA composition (e.g., mRNA)described herein is administered to subjects of age 56-85. In someembodiments, an RNA (e.g., mRNA) composition described herein isadministered (e.g., by intramuscular injection) as a single dose.

In some embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that RBD-specific IgG (e.g.,polyclonal response) induced by such RNA compositions and/or methodsexhibit a higher binding affinity to RBD, as compared to a referencehuman monoclonal antibody with SARS-CoV-2 RBD-binding affinity (e.g.,CR3022 as described in J. ter Meulen et al., PLOS Med. 3, e237 (2006).)

In some embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity across a panel (e.g., at least 10, atleast 15, or more) of SARs-CoV-2 spike variants (e.g., across a panel ofvariants described herein). In some embodiments, such SARs-CoV-2 spikevariants include mutations in RBD (e.g., but not limited to Q321L,V341I, A348T, N354D, S359N, V367F, K378R, R408I, Q409E, A435S, N439K,K458R, I472V, G476S, S477N, V483A, Y508H, H519P, etc., as compared toSEQ ID NO: 1), and/or mutations in spike protein (e.g., but not limitedto D614G, etc., as compared to SEQ ID NO: 1). Those skilled in the artare aware of various spike variants, and/or resources that document them(e.g., the Table of mutating sites in Spike maintained by the COVID-19Viral Genome Analysis Pipeline and found athttps://cov.lanl.gov/components/sequence/COV/int_sites_tbls.comp) (lastaccessed 24 Aug. 2020), and, reading the present specification, willappreciate that RNA (e.g, mRNA) compositions and/or methods describedherein can be characterized for their ability to induce sera invaccinated subject that display neutralizing activity with respect toany or all of such variants and/or combinations thereof.

In particular embodiments, RNA (e.g., mRNA) compositions encoding RBD ofa SARS-CoV-2 spike protein are characterized in that sera of vaccinatedsubjects display neutralizing activity across a panel (e.g., at least10, at least 15, or more) of SARs-CoV-2 spike variants including RBDvariants (e.g., but not limited to Q321L, V341I, A348T, N354D, S359N,V367F, K378R, R408I, Q409E, A435S, N439K, K458R, I472V, G476S, S477N,V483A, Y508H, H519P, etc., as compared to SEQ ID NO: 1) and spikeprotein variants (e.g., but not limited to D614G, as compared to SEQ IDNO: 1).

In particular embodiments, RNA (e.g., mRNA) compositions encoding aSARS-CoV-2 spike protein variant that includes two consecutive prolinesubstitutions at amino acid positions 986 and 987, at the top of thecentral helix in the S2 subunit, are characterized in that sera ofvaccinated subjects display neutralizing activity across a panel (e.g.,at least 10, at least 15, or more) of SARs-CoV-2 spike variantsincluding RBD variants (e.g., but not limited to Q321L, V341I, A348T,N354D, S359N, V367F, K378R, R408I, Q409E, A435S, N439K, K458R, I472V,G476S, S477N, V483A, Y508H, H519P, etc., as compared to SEQ ID NO: 1)and spike protein variants (e.g., but not limited to D614G, as comparedto SEQ ID NO: 1). For example, in some embodiments, an RNA (e.g., mRNA)composition encoding SEQ ID NO: 7 (S P2) elicits an immune responseagainst any one of a SARs-CoV-2 spike variant including RBD variants(e.g., but not limited to Q321L, V341I, A348T, N354D, S359N, V367F,K378R, R408I, Q409E, A435S, N439K, K458R, I472V, G476S, S477N, V483A,Y508H, H519P, etc., as compared to SEQ ID NO: 1) and spike proteinvariants (e.g., but not limited to D614G, as compared to SEQ ID NO: 1).In some embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against one or more SARs-CoV-2 spikevariants including a mutation at position 501 in spike protein ascompared to SEQ ID NO: 1. In some embodiments, RNA (e.g., mRNA)compositions and/or methods described herein are characterized in thatsera of vaccinated subjects display neutralizing activity against one ormore SARs-CoV-2 spike variants including a N501Y mutation in spikeprotein as compared to SEQ ID NO: 1.

Said one or more SARs-CoV-2 spike variants including a mutation atposition 501 in spike protein as compared to SEQ ID NO: 1 or said one ormore SARs-CoV-2 spike variants including a N501Y mutation in spikeprotein as compared to SEQ ID NO: 1 may include one or more furthermutations as compared to SEQ ID NO: 1 (e.g., but not limited to H69/V70deletion, Y144 deletion, A570D, D614G, P681H, T716I, S982A, D1118H,D80A, D215G, E484K, A701V, L18F, R246I, K417N, L242/A243/L244 deletionetc., as compared to SEQ ID NO: 1).

In particular embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against SARs-CoV-2 spike variant “Variantof Concern 202012/01” (VOC-202012/01; also known as lineage B.1.1.7).The variant had previously been named the first Variant UnderInvestigation in December 2020 (VUI—202012/01) by Public Health England,but was reclassified to a Variant of Concern (VOC-202012/01).VOC-202012/01 is a variant of SARS-CoV-2 which was first detected inOctober 2020 during the COVID-19 pandemic in the United Kingdom from asample taken the previous month, and it quickly began to spread bymid-December. It is correlated with a significant increase in the rateof COVID-19 infection in United Kingdom; this increase is thought to beat least partly because of change N501Y inside the spike glycoprotein'sreceptor-binding domain, which is needed for binding to ACE2 in humancells. The VOC-202012/01 variant is defined by 23 mutations: 13non-synonymous mutations, 4 deletions, and 6 synonymous mutations (i.e.,there are 17 mutations that change proteins and six that do not). Thespike protein changes in VOC 202012/01 include deletion 69-70, deletion144, N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H. One of themost important changes in VOC-202012/01 seems to be N501Y, a change fromasparagine (N) to tyrosine (Y) at amino-acid site 501. This mutationalone or in combination with the deletion at positions 69/70 in the Nterminal domain (NTD) may enhance the transmissibility of the virus.

In particular embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against SARs-CoV-2 spike variant includingthe following mutations: deletion 69-70, deletion 144, N501Y, A570D,D614G, P681H, T716I, S982A, and D1118H as compared to SEQ ID NO: 1.

In particular embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against SARs-CoV-2 spike variant “501.V2”.This variant was first observed in samples from October 2020, and sincethen more than 300 cases with the 501.V2 variant have been confirmed bywhole genome sequencing (WGS) in South Africa, where in December 2020 itwas the dominant form of the virus. Preliminary results indicate thatthis variant may have an increased transmissibility. The 501.V2 variantis defined by multiple spike protein changes including: D80A, D215G,E484K, N501Y and A701V, and more recently collected viruses haveadditional changes: L18F, R246I, K417N, and deletion 242-244.

In particular embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against SARs-CoV-2 spike variant includingthe following mutations: D80A, D215G, E484K, N501Y and A701V as comparedto SEQ ID NO: 1, and optionally: L18F, R246I, K417N, and deletion242-244 as compared to SEQ ID NO: 1. Said SARs-CoV-2 spike variant mayalso include a D614G mutation as compared to SEQ ID NO: 1.

In some embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against one or more SARs-CoV-2 spikevariants including a H69/V70 deletion in spike protein as compared toSEQ ID NO: 1.

In some embodiments, one or more SARs-CoV-2 spike variants including aH69/V70 deletion in spike protein as compared to SEQ ID NO: 1 mayinclude one or more further mutations as compared to SEQ ID NO: 1 (e.g.,but not limited to Y144 deletion, N501Y, A570D, D614G, P681H, T716I,S982A, D1118H, D80A, D215G, E484K, A701V, L18F, R246I, K417N,L242/A243/L244 deletion, Y453F, I692V, S1147L, M1229I etc., as comparedto SEQ ID NO: 1), In particular embodiments, RNA (e.g., mRNA)compositions and/or methods described herein are characterized in thatsera of vaccinated subjects display neutralizing activity againstSARs-CoV-2 spike variant “Variant of Concern 202012/01” (VOC-202012/01;also known as lineage B.1.1.7).

In particular embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against SARs-CoV-2 spike variant includingthe following mutations: deletion 69-70, deletion 144, N501Y, A570D,D614G, P681H, T716I, S982A, and D1118H as compared to SEQ ID NO: 1.

In particular embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against SARs-CoV-2 spike variant “Cluster5”, also referred to as ΔFVI-spike by the Danish State Serum Institute(SSI). It was discovered in North Jutland, Denmark, and is believed tohave been spread from minks to humans via mink farms. In cluster 5,several different mutations in the spike protein of the virus have beenconfirmed. The specific mutations include 69-70deltaHV (a deletion ofthe histidine and valine residues at the 69th and 70th position in theprotein), Y453F (a change from tyrosine to phenylalanine at position453), I692V (isoleucine to valine at position 692), M1229I (methionineto isoleucine at position 1229), and optionally S1147L (serine toleucine at position 1147).

In particular embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against SARs-CoV-2 spike variant includingthe following mutations: deletion 69-70, Y453F, I692V, M1229I, andoptionally S1147L, as compared to SEQ ID NO: 1.

In some embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against one or more SARs-CoV-2 spikevariants including a mutation at position 614 in spike protein ascompared to SEQ ID NO: 1. In some embodiments, RNA (e.g., mRNA)compositions and/or methods described herein are characterized in thatsera of vaccinated subjects display neutralizing activity against one ormore SARs-CoV-2 spike variants including a D614G mutation in spikeprotein as compared to SEQ ID NO: 1.

In some embodiments, one or more SARs-CoV-2 spike variants including amutation at position 614 in spike protein as compared to SEQ ID NO: 1 orsaid one or more SARs-CoV-2 spike variants including a D614G mutation inspike protein as compared to SEQ ID NO: 1 may include one or morefurther mutations as compared to SEQ ID NO: 1 (e.g., but not limited toH69/V70 deletion, Y144 deletion, N501Y, A570D, P681H, T716I, S982A,D1118H, D80A, D215G, E484K, A701V, L18F, R246I, K417N, L242/A243/L244deletion, Y453F, I692V, S1147L, M1229I etc., as compared to SEQ ID NO:1).

In particular embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against SARs-CoV-2 spike variant “Variantof Concern 202012/01” (VOC-202012/01; also known as lineage B.1.1.7).

In particular embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against SARs-CoV-2 spike variant includingthe following mutations: deletion 69-70, deletion 144, N501Y, A570D,D614G, P681H, T716I, S982A, and D1118H as compared to SEQ ID NO: 1.

In particular embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against SARs-CoV-2 spike variant includingthe following mutations: D80A, D215G, E484K, N501Y, A701V, and D614G ascompared to SEQ ID NO: 1, and optionally: L18F, R246I, K417N, anddeletion 242-244 as compared to SEQ ID NO: 1.

In some embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against one or more SARs-CoV-2 spikevariants including a mutation at positions 501 and 614 in spike proteinas compared to SEQ ID NO: 1. In some embodiments, RNA (e.g., mRNA)compositions and/or methods described herein are characterized in thatsera of vaccinated subjects display neutralizing activity against one ormore SARs-CoV-2 spike variants including a N501Y mutation and a D614Gmutation in spike protein as compared to SEQ ID NO: 1.

In some embodiments, one or more SARS-CoV-2 spike variants including amutation at positions 501 and 614 in spike protein as compared to SEQ IDNO: 1 or said one or more SARs-CoV-2 spike variants including a N501Ymutation and a D614G mutation in spike protein as compared to SEQ ID NO:1 may include one or more further mutations as compared to SEQ ID NO: 1(e.g., but not limited to H69/V70 deletion, Y144 deletion, A570D, P681H,T716I, S982A, D1118H, D80A, D215G, E484K, A701V, L18F, R246I, K417N,L242/A243/L244 deletion, Y453F, I692V, S1147L, M1229I etc., as comparedto SEQ ID NO: 1).

In particular embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against SARs-CoV-2 spike variant “Variantof Concern 202012/01” (VOC-202012/01; also known as lineage B.1.1.7).

In particular embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against SARs-CoV-2 spike variant includingthe following mutations: deletion 69-70, deletion 144, N501Y, A570D,D614G, P681H, T716I, S982A, and D1118H as compared to SEQ ID NO: 1.

In particular embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against SARs-CoV-2 spike variant includingthe following mutations: D80A, D215G, E484K, N501Y, A701V, and D614G ascompared to SEQ ID NO: 1, and optionally: L18F, R246I, K417N, anddeletion 242-244 as compared to SEQ ID NO: 1.

In some embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against one or more SARs-CoV-2 spikevariants including a mutation at position 484 in spike protein ascompared to SEQ ID NO: 1. In some embodiments, RNA (e.g., mRNA)compositions and/or methods described herein are characterized in thatsera of vaccinated subjects display neutralizing activity against one ormore SARs-CoV-2 spike variants including a E484K mutation in spikeprotein as compared to SEQ ID NO: 1.

In some embodiments, one or more SARs-CoV-2 spike variants including amutation at position 484 in spike protein as compared to SEQ ID NO: 1 orsaid one or more SARs-CoV-2 spike variants including a E484K mutation inspike protein as compared to SEQ ID NO: 1 may include one or morefurther mutations as compared to SEQ ID NO: 1 (e.g., but not limited toH69/V70 deletion, Y144 deletion, N501Y, A570D, D614G, P681H, T716I,S982A, D1118H, D80A, D215G, A701V, L18F, R246I, K417N, L242/A243/L244deletion, Y453F, I692V, S1147L, M1229I, T20N, P26S, D138Y, R190S, K417T,H655Y, T1027I, V1176F etc., as compared to SEQ ID NO: 1).

In particular embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against SARs-CoV-2 spike variant “501.V2”.

In particular embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against SARs-CoV-2 spike variant includingthe following mutations: D80A, D215G, E484K, N501Y, and A701V, ascompared to SEQ ID NO: 1, and optionally: L18F, R246I, K417N, anddeletion 242-244 as compared to SEQ ID NO: 1. Said SARs-CoV-2 spikevariant may also include a D614G mutation as compared to SEQ ID NO: 1.

Lineage B.1.1.248, known as the Brazil(ian) variant, is one of thevariants of SARS-CoV-2 which has been named P.1 lineage and has 17unique amino acid changes, 10 of which in its spike protein, includingN501Y and E484K. B.1.1.248 originated from B.1.1.28. E484K is present inboth B.1.1.28 and B.1.1.248. B.1.1.248 has a number of S-proteinpolymorphisms [L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y,H655Y, T1027I, V1176F] and is similar in certain key RBD positions(K417, E484, N501) to variant described from South Africa.

In particular embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against SARs-CoV-2 spike variant“B.1.1.28”.

In particular embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against SARs-CoV-2 spike variant“B.1.1.248”.

In particular embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against SARs-CoV-2 spike variant includingthe following mutations: L18F, T20N, P26S, D138Y, R190S, K417T, E484K,N501Y, H655Y, T1027I, and V1176F as compared to SEQ ID NO: 1.

In some embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against one or more SARs-CoV-2 spikevariants including a mutation at positions 501 and 484 in spike proteinas compared to SEQ ID NO: 1. In some embodiments, RNA (e.g., mRNA)compositions and/or methods described herein are characterized in thatsera of vaccinated subjects display neutralizing activity against one ormore SARs-CoV-2 spike variants including a N501Y mutation and a E484Kmutation in spike protein as compared to SEQ ID NO: 1.

In some embodiments, one or more SARs-CoV-2 spike variants including amutation at positions 501 and 484 in spike protein as compared to SEQ IDNO: 1 or said one or more SARs-CoV-2 spike variants including a N501Ymutation and a E484K mutation in spike protein as compared to SEQ ID NO:1 may include one or more further mutations as compared to SEQ ID NO: 1(e.g., but not limited to H69/V70 deletion, Y144 deletion, A570D, D614G,P681H, T716I, S982A, D1118H, D80A, D215G, A701V, L18F, R246I, K417N,L242/A243/L244 deletion, Y453F, I692V, S1147L, M1229I, T20N, P26S,D138Y, R190S, K417T, H655Y, T1027I, V1176F etc., as compared to SEQ IDNO: 1).

In particular embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against SARs-CoV-2 spike variant “501.V2”.

In particular embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against SARs-CoV-2 spike variant includingthe following mutations: D80A, D215G, E484K, N501Y and A701V as comparedto SEQ ID NO: 1, and optionally: L18F, R246I, K417N, and deletion242-244 as compared to SEQ ID NO: 1. Said SARs-CoV-2 spike variant mayalso include a D614G mutation as compared to SEQ ID NO: 1.

In particular embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against SARs-CoV-2 spike variant“B.1.1.248”.

In particular embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against SARs-CoV-2 spike variant includingthe following mutations: L18F, T20N, P26S, D138Y, R190S, K417T, E484K,N501Y, H655Y, T1027I, and V1176F as compared to SEQ ID NO: 1.

In some embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against one or more SARs-CoV-2 spikevariants including a mutation at positions 501, 484 and 614 in spikeprotein as compared to SEQ ID NO: 1. In some embodiments, RNA (e.g.,mRNA) compositions and/or methods described herein are characterized inthat sera of vaccinated subjects display neutralizing activity againstone or more SARs-CoV-2 spike variants including a N501Y mutation, aE484K mutation and a D614G mutation in spike protein as compared to SEQID NO: 1.

In some embodiments, one or more SARs-CoV-2 spike variants including amutation at positions 501, 484 and 614 in spike protein as compared toSEQ ID NO: 1 or said one or more SARs-CoV-2 spike variants including aN501Y mutation, a E484K mutation and a D614G mutation in spike proteinas compared to SEQ ID NO: 1 may include one or more further mutations ascompared to SEQ ID NO: 1 (e.g., but not limited to H69/V70 deletion,Y144 deletion, A570D, P681H, T716I, S982A, D1118H, D80A, D215G, A701V,L18F, R246I, K417N, L242/A243/L244 deletion, Y453F, I692V, S1147L,M1229I, T20N, P26S, D138Y, R190S, K417T, H655Y, T1027I, V1176F etc., ascompared to SEQ ID NO: 1).

In particular embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against SARs-CoV-2 spike variant includingthe following mutations: D80A, D215G, E484K, N501Y, A701V, and D614G ascompared to SEQ ID NO: 1, and optionally: L18F, R246I, K417N, anddeletion 242-244 as compared to SEQ ID NO: 1.

In some embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against one or more SARs-CoV-2 spikevariants including a L242/A243/L244 deletion in spike protein ascompared to SEQ ID NO: 1.

In some embodiments, one or more SARs-CoV-2 spike variants including aL242/A243/L244 deletion in spike protein as compared to SEQ ID NO: 1 mayinclude one or more further mutations as compared to SEQ ID NO: 1 (e.g.,but not limited to H69/V70 deletion, Y144 deletion, N501Y, A570D, D614G,P681H, T716I, S982A, D1118H, D80A, D215G, E484K, A701V, L18F, R246I,K417N, Y453F, I692V, S1147L, M1229I, T20N, P26S, D138Y, R190S, K417T,H655Y, T1027I, V1176F etc., as compared to SEQ ID NO: 1).

In particular embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against SARs-CoV-2 spike variant “501.V2”.

In particular embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against SARs-CoV-2 spike variant includingthe following mutations: D80A, D215G, E484K, N501Y, A701V and deletion242-244 as compared to SEQ ID NO: 1, and optionally: L18F, R246I, andK417N, as compared to SEQ ID NO: 1. Said SARs-CoV-2 spike variant mayalso include a D614G mutation as compared to SEQ ID NO: 1.

In some embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against one or more SARs-CoV-2 spikevariants including a mutation at position 417 in spike protein ascompared to SEQ ID NO: 1. In some embodiments, RNA (e.g., mRNA)compositions and/or methods described herein are characterized in thatsera of vaccinated subjects display neutralizing activity against one ormore SARs-CoV-2 spike variants including a K417N or K417T mutation inspike protein as compared to SEQ ID NO: 1.

In some embodiments, one or more SARs-CoV-2 spike variants including amutation at position 417 in spike protein as compared to SEQ ID NO: 1 orsaid one or more SARs-CoV-2 spike variants including a K417N or K417Tmutation in spike protein as compared to SEQ ID NO: 1 may include one ormore further mutations as compared to SEQ ID NO: 1 (e.g., but notlimited to H69/V70 deletion, Y144 deletion, N501Y, A570D, D614G, P681H,T716I, S982A, D1118H, D80A, D215G, E484K, A701V, L18F, R246I,L242/A243/L244 deletion, Y453F, I692V, S1147L, M1229I, T20N, P26S,D138Y, R190S, H655Y, T1027I, V1176F etc., as compared to SEQ ID NO: 1).

In particular embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against SARs-CoV-2 spike variant “501.V2”.

In particular embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against SARs-CoV-2 spike variant includingthe following mutations: D80A, D215G, E484K, N501Y, A701V and K417N, ascompared to SEQ ID NO: 1, and optionally: L18F, R246I, and deletion242-244 as compared to SEQ ID NO: 1. Said SARs-CoV-2 spike variant mayalso include a D614G mutation as compared to SEQ ID NO: 1.

In particular embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against SARs-CoV-2 spike variant“B.1.1.248”.

In particular embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against SARs-CoV-2 spike variant includingthe following mutations: L18F, T20N, P26S, D138Y, R190S, K417T, E484K,N501Y, H655Y, T1027I, and V1176F as compared to SEQ ID NO: 1.

In some embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against one or more SARs-CoV-2 spikevariants including a mutation at positions 417 and 484 and/or 501 inspike protein as compared to SEQ ID NO: 1. In some embodiments, RNA(e.g., mRNA) compositions and/or methods described herein arecharacterized in that sera of vaccinated subjects display neutralizingactivity against one or more SARs-CoV-2 spike variants including a K417Nor K417T mutation and a E484K and/or N501Y mutation in spike protein ascompared to SEQ ID NO: 1.

In some embodiments, one or more SARs-CoV-2 spike variants including amutation at positions 417 and 484 and/or 501 in spike protein ascompared to SEQ ID NO: 1 or said one or more SARs-CoV-2 spike variantsincluding a K417N or K417T mutation and a E484K and/or N501Y mutation inspike protein as compared to SEQ ID NO: 1 may include one or morefurther mutations as compared to SEQ ID NO: 1 (e.g., but not limited toH69/V70 deletion, Y144 deletion, A570D, D614G, P681H, T716I, S982A,D1118H, D80A, D215G, A701V, L18F, R246I, L242/A243/L244 deletion, Y453F,I692V, S1147L, M1229I, T20N, P26S, D138Y, R190S, H655Y, T1027I, V1176Fetc., as compared to SEQ ID NO: 1).

In particular embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against SARs-CoV-2 spike variant “501.V2”.

In particular embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against SARs-CoV-2 spike variant includingthe following mutations: D80A, D215G, E484K, N501Y, A701V and K417N, ascompared to SEQ ID NO: 1, and optionally: L18F, R246I, and deletion242-244 as compared to SEQ ID NO: 1. Said SARs-CoV-2 spike variant mayalso include a D614G mutation as compared to SEQ ID NO: 1.

In particular embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against SARs-CoV-2 spike variant“B.1.1.248”.

In particular embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against SARs-CoV-2 spike variant includingthe following mutations: L18F, T20N, P26S, D138Y, R190S, K417T, E484K,N501Y, H655Y, T1027I, and V1176F as compared to SEQ ID NO: 1.

In some embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against SARs-CoV-2 spike variant of theOmicron (B.1.1.529) variant. Omicron (B.1.1.529) variant is a variant ofSARS-CoV-2 which was detected in South Africa. Multiple Omicron variantsor sublineages have arisen, including e.g., the BA.1, BA.2, BA.2.12.1,BA.3, BA.4, BA.5, and BA.2.75 sublineages. As used herein, unlessotherwise specified, “Omicron variant” refers to the first disclosedOmicron variant (BA.1) or any variant thereof that has since arisen(e.g., Omicron variants described herein). In some embodiments, thespike protein changes in Omicron (B.1.1.529) BA.1 variant include A67V,Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE (insertion of EPEfollowing amino acid 214), G339D, S371L, S373P, S375F, K417N, N440K,G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K,D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, andL981F. In some embodiments, the spike protein changes in Omicron(B.1.1.529) variant include A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211,L212I, ins214EPE (insertion of EPE following amino acid 214), G339D,S371L, S373P, S375F, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y,Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H,N969K, and L981F. In some embodiments, the spike changes in Omicron BA.2variant include T19I, Δ24-26, A27S, G142D, V213G, G339D, S371F, S373P,S375F, T376A, D405N, R408S, K417N, N440K, S477N, T478K, E484A, Q493R,Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H,N969K. In some embodiments BA.4 and BA.5 have the same Spike proteinamino acid sequence, in which case “BA.4/5” is used to either Omicronvariant. In some embodiments, the spike changes in Omicron BA.4/5include: T19I, Δ24-26, A27S, Δ69/70, G142D, V213G, G339D, S371F, S373P,S375F, T376A, D405N, R408S, K417N, N440K, L452R, 5477N, T478K, E484A,F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y,Q954H, and N969K. In some embodiments, the spike changes in OmicronBA.2.75 include T19I, Δ24-26, A27S, G142D, K147E, W152R, F157L, I210V,V213G, G257S, G339H, N354D, S371F, S373P, S375F, T376A, D405N, R408S,K417N, N440K, G446S, N460K, S477N, T478K, E484A, Q498R, N501Y, Y505HD614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K.

In some embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against SARs-CoV-2 spike variant includingat least 10, at least 15, at least 20, at least 21, at least 22, atleast 23, at least 24, at least 25, at least 26, at least 27, at least28, at least 29, at least 30, at least 31, at least 32, at least 33, atleast 34, at least 35, at least 36, or at least 37 of the followingmutations: T547K, H655Y, D614G, N679K, P681H, N969K, S373P, S371L,N440K, G339D, G446S, N856K, N764K, K417N, D796Y, Q954H, T95I, A67V,L981F, S477N, G496S, T478K, Q498R, Q493R, E484A, N501Y, S375F, Y505H,V143del, H69del, V70del, N211del, L212I, ins214EPE, G142D, Y144del,Y145del, 1141del, Y144F, Y145D, G142del, as compared to SEQ ID NO: 1.

In some embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against SARs-CoV-2 spike variant includingat least 10, at least 15, at least 20, at least 21, at least 22, atleast 23, at least 24, or all of the following mutations: T547K, H655Y,D614G, N679K, P681H, N969K, S373P, 5371L, N440K, G339D, G446S, N856K,N764K, K417N, D796Y, Q954H, T95I, A67V, L981F, S477N, G496S, T478K,Q498R, Q493R, E484A, as compared to SEQ ID NO: 1. Said SARs-CoV-2 spikevariant may include at least 1, at least 2, at least 3, at least 4, atleast 5, or all of the following mutations: N501Y, S375F, Y505H,V143del, H69del, V70del, as compared to SEQ ID NO: 1, and/or may includeat least 1, at least 2, at least 3, at least 4, at least 5, or all ofthe following mutations: N211del, L212I, ins214EPE, G142D, Y144del,Y145del, as compared to SEQ ID NO: 1. In some embodiments, saidSARs-CoV-2 spike variant may include at least 1, at least 2, at least 3,or all of the following mutations: L141del, Y144F, Y145D, G142del, ascompared to SEQ ID NO: 1.

In some embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against a SARs-CoV-2 spike variantincluding at least 10, at least 15, at least 20, at least 21, at least22, at least 23, at least 24, at least 25, at least 26, at least 27, atleast 28, at least 29, at least 30, at least 31, at least 32, at least33 of the following mutations: A67V, Δ69-70, T95I, G142D, Δ143-145,Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S,S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G,H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F, ascompared to SEQ ID NO: 1.

In some embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against a SARS-CoV-2 spike variantincluding at least 10, at least 15, at least 20, at least 21, at least22, at least 23, at least 24, at least 25, at least 26, at least 27, atleast 28, at least 29, at least 30, or at least 31, of the followingmutations: T19I, Δ24-26, A27S, G142D, V213G, G339D, S371F, S373P, S375F,T376A, D405N, R408S, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R,N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, ascompared to SEQ ID NO: 1.

In some embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against a SARS-CoV-2 spike variantincluding at least 10, at least 15, at least 20, at least 21, at least22, at least 23, at least 24, at least 25, at least 26, at least 27, atleast 28, at least 29, at least 30, at least 31, at least 32, at least33, or at least 34 of the following mutations: T19I, Δ24-26, A27S,Δ69/70, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S,K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H,D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K, as comparedto SEQ ID NO: 1.

In some embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against a SARs-CoV-2 spike variantincluding the following mutations: A67V, Δ69-70, T95I, G142D, Δ143-145,Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S,S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G,H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F, ascompared to SEQ ID NO: 1.

In some embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizating against a SARS-CoV-2 spike variant including thefollowing mutations: T19I, Δ24-26, A27S, G142D, V213G, G339D, S371F,S373P, S375F, T376A, D405N, R408S, K417N, N440K, S477N, T478K, E484A,Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y,Q954H, N969K. In some embodiments, RNA (e.g., mRNA) compositions and/ormethods described herein are characterized in that sera of vaccinatedsubjects display neutralizating against SARS-CoV2 spike variantincluding the following mutations: T19I, Δ24-26, A27S, Δ69/70, G142D,V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K,L452R, 5477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y,N679K, P681H, N764K, D796Y, Q954H, and N969K, as compared to SEQ ID NO:1.

In some embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against SARs-CoV-2 spike variant includingthe following mutations: A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211,L212I, ins214EPE, G339D, S371L, S373P, S375F, S477N, T478K, E484A,Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H,N764K, D796Y, N856K, Q954H, N969K, and L981F, as compared to SEQ ID NO:1.

In some embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein are characterized in that sera of vaccinated subjectsdisplay neutralizing activity against SARs-CoV-2 spike variant includingthe following mutations: T19I, Δ24-26, A27S, G142D, K147E, W152R, F157L,I210V, V213G, G257S, G339H, N354D, S371F, S373P, S375F, T376A, D405N,R408S, K417N, N440K, G446S, N460K, S477N, T478K, E484A, Q498R, N501Y,Y505H D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K, ascompared to SEQ ID NO: 1.

SARs-CoV-2 spike proteins encoded by RNA described herein may or may notinclude a D614G mutation as compared to SEQ ID NO: 1.

In some embodiments, SARS-CoV-2 spike proteins encoded by RNA describedherein comprise a mutation in a furin cleavage site (e.g., in someembodiments residues 682-685 of SEQ ID NO: 1). In some embodiments,SARS-CoV-2 spike proteins encoded by RNA described herein comprise amutation in the furin cleavage site that prevents cleavage by a furinprotease (e.g., a human furin protease). In some embodiments, aSARS-CoV-2 protein described herein comprises a furin mutation disclosedin WO2021163365 or WO2021243122 (e.g., a GSAS mutation), the contents ofboth of which are incorporated by reference herein in their entirety.

In some embodiments, RNA (e.g., mRNA) compositions and/or methodsdescribed herein can provide protection against SARS-CoV-2 and/ordecrease severity of SARS-CoV-2 infection in at least 50% of subjectsreceiving such RNA compositions and/or methods.

In some embodiments, populations to be treated with RNA (e.g., mRNA)compositions described herein include subjects of age 18-55. In someembodiments, populations to be treated with RNA (e.g., mRNA)compositions described herein include subjects of age 56-85. In someembodiments, populations to be treated with RNA (e.g., mRNA)compositions described herein include older subjects (e.g., over age 60,65, 70, 75, 80, 85, etc, for example subjects of age 65-85). In someembodiments, populations to be treated with RNA (e.g., mRNA)compositions described herein include subjects of age 18-85. In someembodiments, populations to be treated with RNA (e.g., mRNA)compositions described herein include subjects of age 18 or younger. Insome embodiments, populations to be treated with RNA (e.g., mRNA)compositions described herein include subjects of age 12 or younger. Insome embodiments, populations to be treated with RNA (e.g., mRNA)compositions described herein include subjects of age 10 or younger. Insome embodiments, populations to be treated with RNA (e.g., mRNA)compositions described herein may include adolescent populations (e.g.,individuals approximately 12 to approximately 17 years of age). In someembodiments, populations to be treated with RNA (e.g., mRNA)compositions described herein may include pediatric populations (e.g.,as described herein). In some embodiments, populations to be treatedwith RNA (e.g., mRNA) compositions described herein include infants(e.g., less than 1 year old). In some embodiments, populations to betreated with RNA (e.g., mRNA) compositions described herein do notinclude infants (e.g., less than 1 year) whose mothers have receivedsuch RNA (e.g., mRNA) compositions described herein during pregnancy.Without wishing to be bound by any particular theory, a rat study hassuggested that a SARS-CoV-2 neutralizing antibody response induced infemale rats given such RNA compositions during pregnancy can pass ontofetuses. In some embodiments, populations to be treated with RNA (e.g.,mRNA) compositions described herein include infants (e.g., less than 1year) whose mothers did not receive such RNA compositions describedherein during pregnancy. In some embodiments, populations to be treatedwith RNA (e.g., mRNA) compositions described herein may include pregnantwomen; in some embodiments, infants whose mothers were vaccinated duringpregnancy (e.g., who received at least one dose, or alternatively onlywho received both doses), are not vaccinated during the first weeks,months, or even years (e.g., 1, 2, 3, 4, 5, 6, 7, 8 weeks or more, or 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24 months or more, or 1, 2, 3, 4, 5 years or more) post-birth.Alternatively or additionally, in some embodiments, infants whosemothers were vaccinated during pregnancy (e.g., who received at leastone dose, or alternatively only who received both doses), receivereduced vaccination (e.g., lower doses and/or smaller numbers ofadministrations—e.g., boosters—and/or lower total exposure over a givenperiod of time) after birth, for example during the first weeks, months,or even years (e.g., 1, 2, 3, 4, 5, 6, 7, 8 weeks or more, or 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24 months or more, or 1, 2, 3, 4, 5 years or more) post-birth or mayneed reduced vaccination (e.g., lower doses and/or smaller numbers ofadministrations—e.g., boosters—over a given period of time), In someembodiments, compositions as provided herein are administered topopulations that do not include pregnant women.

In some particular embodiments, compositions as provided herein areadministered to pregnant women according to a regimen that includes afirst dose administered after about 24 weeks of gestation (e.g., afterabout 22, 23, 24, 25, 26, 27, 28 or more weeks of gestation); in someembodiments, compositions as provided herein are administered topregnant women according to a regimen that includes a first doseadministered before about 34 weeks of gestation (e.g., before about 30,31, 32, 33, 34, 35, 36, 37, 38 weeks of gestation). In some embodiments,compositions as provided herein are administered to pregnant womenaccording to a regimen that includes a first dose administered afterabout 24 weeks (e.g., after about 27 weeks of gestation, e.g., betweenabout 24 weeks and 34 weeks, or between about 27 weeks and 34 weeks) ofgestation and a second dose administered about 21 days later; in someembodiments both doses are administered prior to delivery. Withoutwishing to be bound by any particular theory, it is proposed that such aregimen (e.g., involving administration of a first dose after about 24weeks, or 27 weeks of gestation and optionally before about 34 weeks ofgestation), and optionally a second dose within about 21 days, ideallybefore delivery, may have certain advantages in terms of safety (e.g.,reduced risk of premature delivery or of fetal morbidity or mortality)and/or efficacy (e.g., carryover vaccination imparted to the infant)relative to alternative dosing regimens (e.g., dosing at any time duringpregnancy, refraining from dosing during pregnancy, and/or dosing laterin pregnancy for example so that only one dose is administered duringgestation. In some embodiments, infants born of mothers vaccinatedduring pregnancy, e.g., according to a particular regimen as describedherein, may not need further vaccination, or may need reducedvaccination (e.g., lower doses and/or smaller numbers ofadministrations—e.g., boosters —, and/or lower overall exposure over agiven period of time), for a period of time (e.g., as noted herein)after birth.

In some embodiments, compositions as provided herein are administered topopulations in which women are advised against becoming pregnant for aperiod of time after receipt of the vaccine (e.g., after receipt of afirst dose of the vaccine, after receipt of a final dose of the vaccine,etc.); in some such embodiments, the period of time may be at least 1week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9weeks, at least 10 weeks or more, or may be at least 1 month, at least 2months, at least 3 months, at least 4 months, at least 5 months, atleast 6 months, or more.

In some embodiments, populations to be treated with RNA (e.g., mRNA)compositions described herein may include one or more populations withone or more particularly high risk conditions or history, e.g., as notedherein. For example, in some embodiments, populations to be treated withRNA compositions described herein may include subjects whose professionand/or environmental exposure may dramatically increase their risk ofgetting SARS-CoV-2 infection (including, e.g., but not limited to masstransportation, prisoners, grocery store workers, residents in long-termcare facilities, butchers or other meat processing workers, healthcareworkers, and/or first responders, e.g., emergency responders). Inparticular embodiments, populations to be treated with RNA (e.g., mRNA)compositions described herein may include healthcare workers and/orfirst responders, e.g., emergency responders.

In some embodiments, populations to be treated with RNA (e.g., mRNA)compositions described herein may include those with a history ofsmoking or vaping (e.g., within 6 months, 12 months or more, including ahistory of chronic smoking or vaping). In some embodiments, populationsto be treated with RNA (e.g., mRNA) compositions described herein mayinclude certain ethnic groups that have been determined to be moresusceptible to SARS-CoV-2 infection.

In some embodiments, populations to be treated with RNA (e.g., mRNA)compositions described herein may include certain populations with ablood type that may have been determined to more susceptible toSARS-CoV-2 infection. In some embodiments, populations to be treatedwith RNA (e.g., mRNA) compositions described herein may includeimmunocompromised subjects (e.g., those with HIV/AIDS; cancer patients(e.g., receiving antitumor treatment); patients who are taking certainimmunosuppressive drugs (e.g., transplant patients, cancer patients,etc.); autoimmune diseases or other physiological conditions expected towarrant immunosuppressive therapy (e.g., within 3 months, within 6months, or more); and those with inherited diseases that affect theimmune system (e.g., congenital agammaglobulinemia, congenital IgAdeficiency)). In some embodiments, populations to be treated with RNA(e.g., mRNA) compositions described herein may include those with aninfectious disease. For example, in some embodiments, populations to betreated with RNA (e.g., mRNA) compositions described herein may includethose infected with human immunodeficiency virus (HIV) and/or ahepatitis virus (e.g., HBV, HCV). In some embodiments, populations to betreated with RNA (e.g., mRNA) compositions described herein may includethose with underlying medical conditions. Examples of such underlyingmedical conditions may include, but are not limited to hypertension,cardiovascular disease, diabetes, chronic respiratory disease, e.g.,chronic pulmonary disease, asthma, etc., cancer, and other chronicdiseases such as, e.g., lupus, rheumatoid arthritis, chronic liverdiseases, chronic kidney diseases (e.g., Stage 3 or worse such as insome embodiments as characterized by a glomerular filtration rate (GFR)of less than 60 mL/min/1.73 m²). In some embodiments, populations to betreated with RNA (e.g., mRNA) compositions described herein may includeoverweight or obese subjects, e.g., specifically including those with abody mass index (BMI) above about 30 kg/m². In some embodiments,populations to be treated with RNA (e.g., mRNA) compositions describedherein may include subjects who have prior diagnosis of COVID-19 orevidence of current or prior SARS-CoV-2 infection, e.g., based onserology or nasal swab. In some embodiments, populations to be treatedinclude white and/or non-Hispanic/non-Latino.

In some embodiments, certain RNA (e.g., mRNA) compositions describedherein may be selected for administration to Asian populations (e.g.,Chinese populations), or in particular embodiments to older Asianpopulations (e.g., 60 years old or over, e.g., 60-85 or 65-85 yearsold).

In some embodiments, an RNA (e.g., mRNA) composition as provided hereinis administered to and/or assessed in subject(s) who have beendetermined not to show evidence of prior infection, and/or of presentinfection, before administration; in some embodiments, evidence of priorinfection and/or of present infection, may be or include evidence ofintact virus, or any viral nucleic acid, protein, lipid etc. present inthe subject (e.g., in a biological sample thereof, such as blood, cells,mucus, and/or tissue), and/or evidence of a subject's immune response tothe same. In some embodiments, an RNA (e.g., mRNA) composition asprovided herein is administered to and/or assessed in subject(s) whohave been determined to show evidence of prior infection, and/or ofpresent infection, before administration; in some embodiments, evidenceof prior infection and/or of present infection, may be or includeevidence of intact virus, or any viral nucleic acid, protein, lipid etc.present in the subject (e.g., in a biological sample thereof, such asblood, cells, mucus, and/or tissue), and/or evidence of a subject'simmune response to the same. In some embodiments, a subject isconsidered to have a prior infection based on having a positiveN-binding antibody test result or positive nucleic acid amplificationtest (NAAT) result on the day of Dose 1.

In some embodiments, an RNA (e.g., mRNA) composition as provided hereinis administered to a subject who has been informed of a risk of sideeffects that may include one or more of, for example: chills, fever,headache, injection site pain, muscle pain, tiredness; in someembodiments, an RNA (e.g., mRNA) composition is administered to asubject who has been invited to notify a healthcare provider if one ormore such side effects occurs, is experienced as more than mild ormoderate, persists for a period of more than a day or a few days, or ifany serious or unexpected event is experienced that the subjectreasonably considers may be associated with receipt of the composition.In some embodiments, an RNA (e.g., mRNA) composition as provided hereinis administered to a subject who has been invited to notify a healthcareprovider of particular medical conditions which may include, forexample, one or more of allergies, bleeding disorder or taking a bloodthinner medication, breastfeeding, fever, immunocompromised state ortaking medication that affects the immune system, pregnancy or plan tobecome pregnant, etc. In some embodiments, an RNA (e.g., mRNA)composition as provided herein is administered to a subject who has beeninvited to notify a healthcare provider of having received anotherCOVID-19 vaccine. In some embodiments, an RNA (e.g., mRNA) compositionas provided herein is administered to a subject not having one of thefollowing medical conditions: experiencing febrile illness, receivingimmunosuppressant therapy, receiving anticoagulant therapy, sufferingfrom a bleeding disorder (e.g., one that would contraindicateintramuscular injection), or pregnancy and/or breastfeeding/lactation.In some embodiments, an RNA (e.g., mRNA) composition as provided hereinis administered to a subject not having received another COVID-19vaccine. In some embodiments, an RNA (e.g., mRNA) composition asprovided herein is administered to a subject who has not had an allergicreaction to any component of the RNA (e.g., mRNA) composition. Examplesof such allergic reaction may include, but are not limited to difficultybreathing, swelling of fact and/or throat, fast heartbeat, rash,dizziness and/or weakness. In some embodiments, an RNA (e.g., mRNA)composition as provided herein is administered to a subject who receiveda first dose and did not have an allergic reaction (e.g., as describedherein) to the first dose. In some embodiments where allergic reactionoccurs in subject(s) after receiving a dose of an RNA (e.g., mRNA)composition as provided herein, such subject(s) may be administered oneor more interventions such as treatment to manage and/or reducesymptom(s) of such allergic reactions, for example, fever-reducingand/or anti-inflammatory agents.

In some embodiments, a subject who has received at least one dose of anRNA (e.g., mRNA) composition as provided herein is informed of avoidingbeing exposed to a coronavirus (e.g., SARS-CoV-2) unless and untilseveral days (e.g., at least 7 days, at least 8 days, 9 days, at least10 days, at least 11 days, at least 12 days, at least 13 days, at least14 days, etc.) have passed since administration of a second dose. Forexample, a subject who has received at least one dose of an RNA (e.g.,mRNA) composition as provided herein is informed of taking precautionarymeasures against SARS-CoV-2 infection (e.g., remaining socially distant,wearing masks, frequent hand-washing, etc.) unless and until severaldays (e.g., at least 7 days, at least 8 days, 9 days, at least 10 days,at least 11 days, at least 12 days, at least 13 days, at least 14 days,etc.) have passed since administration of a second dose. Accordingly, insome embodiments, methods of administering an RNA (e.g., mRNA)composition as provided herein comprise administering a second dose ofsuch an RNA (e.g., mRNA) composition as provided herein to a subject whoreceived a first dose and took precautionary measures to avoid beingexposed to a coronavirus (e.g., SARS-CoV-2).

In some embodiments, RNA (e.g., mRNA) compositions described herein maybe delivered to a draining lymph node of a subject in need thereof, forexample, for vaccine priming. In some embodiments, such delivery may beperformed by intramuscular administration of a provided RNA (e.g., mRNA)composition.

In some embodiments, different particular RNA (e.g., mRNA) compositionsmay be administered to different subject population(s); alternatively oradditionally, in some embodiments, different dosing regimens may beadministered to different subject populations. For example, in someembodiments, RNA (e.g., mRNA) compositions administered to particularsubject population(s) may be characterized by one or more particulareffects (e.g., incidence and/or degree of effect) in those subjectpopulations. In some embodiments, such effect(s) may be or comprise, forexample titer and/or persistence of neutralizing antibodies and/or Tcells (e.g., T_(H)1-type Tcells such as CD4⁺ and/or CD8⁺ T cells),protection against challenge (e.g., via injection and/or nasal exposure,etc), incidence, severity, and/or persistence of side effects (e.g.,reactogenicity), etc.

In some embodiments, one or more RNA (e.g., mRNA) compositions describedherein may be administered according to a regimen established to reduceCOVID-19 incidence per 1000 person-years, e.g., based on a laboratorytest such as nucleic acid amplification test (NAAT). In someembodiments, one or more RNA (e.g., mRNA) compositions described hereinmay be administered according to a regimen established to reduceCOVID-19 incidence per 1000 person-years based on a laboratory test suchas nucleic acid amplification test (NAAT) in subjects receiving at leastone dose of a provided RNA (e.g., mRNA) composition with no serologicalor virological evidence (e.g., up to 7 days after receipt of the lastdose) of past SARS-CoV-2 infection. In some embodiments, one or more RNA(e.g., mRNA) compositions described herein may be administered accordingto a regimen established to reduce confirmed severe COVID-19 incidenceper 1000 person-years. In some embodiments, one or more RNA (e.g., mRNA)compositions described herein may be administered according to a regimenestablished to reduce confirmed severe COVID-19 incidence per 1000person-years in subjects receiving at least one dose of a provided RNA(e.g., mRNA) composition with no serological or virological evidence ofpast SARS-CoV-2 infection.

In some embodiments, one or more RNA (e.g., mRNA) compositions describedherein may be administered according to a regimen established to produceneutralizing antibodies directed to a SARS-CoV-2 spike polypeptideand/or an immunogenic fragment thereof (e.g., RBD) as measured in serumfrom a subject that achieves or exceeds a reference level (e.g., areference level determined based on human SARS-CoV-2 infection/COVID-19convalescent sera) for a period of time and/or induction ofcell-mediated immune response (e.g., a T cell response againstSARS-CoV-2), including, e.g., in some embodiments induction of T cellsthat recognize at least one or more MHC-restricted (e.g., MHC classI-restricted) epitopes within a SARS-CoV-2 spike polypeptide and/or animmunogenic fragment thereof (e.g., RBD) for a period of time. In somesuch embodiments, the period of time may be at least 2 months, 3 months,at least 4 months, at least 5 months, at least 6 months, at least 7months, at least 8 months, at least 9 months, at least 10 months, atleast 11 months, at least 12 months or longer. In some embodiments, oneor more epitopes recognized by vaccine-induced T cells (e.g., CD8+ Tcells) may be presented on a MHC class I allele that is present in atleast 50% of subjects in a population, including, e.g., at least 60%, atleast 70%, at least 80%, at least 90%, or more; in some suchembodiments, the MHC class I allele may be HLA-B*0702, HLA-A*2402,HLA-B*3501, HLA-B*4401, or HLA-A*0201. In some embodiments, an epitopemay comprise HLA-A*0201 YLQPRTFLL (SEQ ID NO: 138); HLA-A*0201 RLQSLQTYV(SEQ ID NO: 139); HLA-A*2402 QYIKWPWYI (SEQ ID NO: 140); HLA-A*2402NYNYLYRLF (SEQ ID NO: 141); HLA-A*2402 KWPWYIWLGF (SEQ ID NO: 142);HLA-B*3501 QPTESIVRF (SEQ ID NO: 143); HLA-B*3501 IPFAMQMAY (SEQ ID NO:144); or HLA-B*3501 LPFNDGVYF (SEQ ID NO: 145).

In some embodiments, efficacy is assessed as COVID-19 incidence per 1000person-years in individuals without serological or virological evidenceof past SARS-CoV-2 infection before and during vaccination regimen;alternatively or additionally, in some embodiments, efficacy is assessedas COVID-19 incidence per 1000 person-years in subjects with and withoutevidence of past SARS-CoV-2 infection before and during vaccinationregimen. In some such embodiments, such incidence is of COVID-19 casesconfirmed within a specific time period after the final vaccination dose(e.g., a first dose in a single-dose regimen; a second dose in atwo-dose regimen, etc); in some embodiments, such time period may bewithin (i.e., up to and including 7 days) a particular number of days(e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30 days or more). In someembodiments, such time period may be within 7 days or within 14 days orwithin 21 days or within 28 days. In some embodiments, such time periodmay be within 7 days. In some embodiments, such time period may bewithin 14 days.

In some embodiments (e.g., in some embodiments of assessing efficacy), asubject is determined to have experienced COVID-19 infection if one ormore of the following is established: detection of SARS-CoV-2 nucleicacid in a sample from the subject, detection of antibodies thatspecifically recognize SARS-CoV-2 (e.g., a SARS-Co-V-2 spike protein),one or more symptoms of COVID-19 infection, and combinations thereof. Insome such embodiments, detection of SARS-CoV-2 nucleic acid may involve,for example, NAAT testing on a mid-turbinatae swap sample. In some suchembodiments, detection of relevant antibodies may involve serologicaltesting of a blood sample or portion thereof. In some such embodiments,symptoms of COVID-19 infection may be or include: fever, new orincreased cough, new or increased shortness of breath, chills, new orincreased muscle pain, new loss of taste or smell, sore throat,diarrhea, vomiting and combinations thereof. In some such embodiments,symptoms of COVID-19 infection may be or include: fever, new orincreased cough, new or increased shortness of breath, chills, new orincreased muscle pain, new loss of taste or smell, sore throat,diarrhea, vomiting, fatigue, headache, nasal congestion or runny nose,nausea, and combinations thereof. In some such embodiments, a subject isdetermined to have experienced COVID-19 infection if such subject bothhas experienced one such symptom and also has received a positive testfor SARS-CoV-2 nucleic acid or antibodies, or both. In some suchembodiments, a subject is determined to have experienced COVID-19infection if such subject both has experienced one such symptom and alsohas received a positive test for SARS-CoV-2 nucleic acid. In some suchembodiments, a subject is determined to have experienced COVID-19infection if such subject both has experienced one such symptom and alsohas received a positive test for SARS-CoV-2 antibodies.

In some embodiments (e.g., in some embodiments of assessing efficacy), asubject is determined to have experienced severe COVID-19 infection ifsuch subject has experienced one or more of: clinical signs at restindicative or severe systemic illness (e.g., one or more of respiratoryrate at greater than or equal to 30 breaths per minute, heart rate at orabove 125 beats per minute, SpO₂ less than or equal to 93% on room airat sea level or a PaO₂/FiO₂ below 300 m Hg), respiratory failure (e.g.,one or more of needing high-flow oxygen, noninvasive ventilation,mechanical ventilation, ECMO), evidence of shock (systolic bloodpressure below 90 mm Hg, diastolic blood pressure below 60 mm Hg,requiring vasopressors), significant acute renal, hepatic, or neurologicdysfunction, admission to an intensive care unit, death, andcombinations thereof.

In some embodiments, one or more RNA (e.g., mRNA) compositions describedherein may be administered according to a regimen established to reducethe percentage of subjects reporting at least one of the following: (i)one or more local reactions (e.g., as described herein) for up to 7 daysfollowing each dose; (ii) one or more systemic events for up to 7 daysfollowing each dose; (iii) adverse events (e.g., as described herein)from a first dose to 1 month after the last dose; and/or (iv) seriousadverse events (e.g., as described herein) from a first dose to 6 monthsafter the last dose.

In some embodiments, one or more subjects who have received an RNA(e.g., mRNA) composition as described herein may be monitored (e.g., fora period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days or more,including, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks ormore, including for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 months or more, including forexample 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years or more) to assess, forexample, presence of an immune response to component(s) of theadministered composition, evidence of exposure to and/or immune responseto SARS-CoV-2 or another coronavirus, evidence of any adverse event,etc. In some embodiments, monitoring may be via tele-visit.Alternatively or additionally, in some embodiments, monitoring may bein-person.

In some embodiments, a treatment effect conferred by one or more RNA(e.g., mRNA) compositions described herein may be characterized by (i) aSARS-CoV-2 anti-S1 binding antibody level above a pre-determinedthreshold; (ii) a SARS-CoV-2 anti-RBD binding antibody level above apre-determined threshold; and/or (iii) a SARS-CoV-2 serum neutralizingtiter above a threshold level, e.g., at baseline, 1 month, 3 months, 6months, 9 months, 12 months, 18 months, and/or 24 months aftercompletion of vaccination. In some embodiments, anti-S1 binding antibodyand/or anti-RBD binding antibody levels and/or serum neutralizing titersmay be characterized by geometric mean concentration (GMC), geometricmean titer (GMT), or geometric mean fold-rise (GMFR).

In some embodiments, a treatment effect conferred by one or more RNA(e.g., mRNA) compositions described herein may be characterized in thatpercentage of treated subjects showing a SARS-CoV-2 serum neutralizingtiter above a pre-determined threshold, e.g., at baseline, 1 month, 3months, 6 months, 9 months, 12 months, 18 months, and/or 24 months aftercompletion of vaccination, is higher than the percentage of non-treatedsubjects showing a SARS-CoV-2 serum neutralizing titer above such apre-determined threshold (e.g., as described herein). In someembodiments, a serum neutralizing titer may be characterized bygeometric mean concentration (GMC), geometric mean titer (GMT), orgeometric mean fold-rise (GMFR).

In some embodiments, a treatment effect conferred by one or more RNA(e.g., mRNA) compositions described herein may be characterized bydetection of SARS-CoV-2 NVA-specific binding antibody.

In some embodiments, a treatment effect conferred by one or more RNA(e.g., mRNA) compositions described herein may be characterized bySARS-CoV-2 detection by nucleic acid amplification test.

In some embodiments, a treatment effect conferred by one or more RNA(e.g., mRNA) compositions described herein may be characterized byinduction of cell-mediated immune response (e.g., a T cell responseagainst SARS-CoV-2), including, e.g., in some embodiments induction of Tcells that recognize at least one or more MHC-restricted (e.g., MHCclass I-restricted) epitopes within a SARS-CoV-2 spike polypeptideand/or an immunogenic fragment thereof (e.g., RBD). In some embodiments,one or more epitopes recognized by vaccine-induced T cells (e.g., CD8+ Tcells) may be presented on a MHC class I allele that is present in atleast 50% of subjects in a population, including, e.g., at least 60%, atleast 70%, at least 80%, at least 90%, or more; in some suchembodiments, the MHC class I allele may be HLA-B*0702, HLA-A*2402,HLA-B*3501, HLA-B*4401, or HLA-A*0201. In some embodiments, an epitopemay comprise HLA-A*0201 YLQPRTFLL (SEQ ID NO: 138); HLA-A*0201 RLQSLQTYV(SEQ ID NO: 139); HLA-A*2402 QYIKWPWYI (SEQ ID NO: 140); HLA-A*2402NYNYLYRLF (SEQ ID NO: 141); HLA-A*2402 KWPWYIWLGF (SEQ ID NO: 142);HLA-B*3501 QPTESIVRF (SEQ ID NO: 143); HLA-B*3501 IPFAMQMAY (SEQ ID NO:144); or HLA-B*3501 LPFNDGVYF (SEQ ID NO: 145).

In some embodiments, primary vaccine efficacy (VE) of one or more RNA(e.g., mRNA) compositions described herein may be established when thereis sufficient evidence (posterior probability) that either primary VE1or both primary VE1 and primary VE2 are >30% or higher (including, e.g.,greater than 40%, greater than 50%, greater than 60%, greater than 70%,greater than 80%, greater than 90%, greater than 95%, greater than 96%,greater than 97%, greater than 98%, or higher), wherein primary VE isdefined as primary VE=100×(1−IRR); and IRR is calculated as the ratio ofCOVID-19 illness rate in the vaccine group to the corresponding illnessrate in the placebo group. Primary VE1 represents VE for prophylacticRNA (e.g., mRNA) compositions described herein against confirmedCOVID-19 in participants without evidence of infection beforevaccination, and primary VE2 represents VE for prophylactic RNA (e.g.,mRNA) compositions described herein against confirmed COVID-19 in allparticipants after vaccination. In some embodiments, primary VE1 and VE2can be evaluated sequentially to control the overall type I error of2.5% (hierarchical testing). In some embodiments where one or more RNA(e.g., mRNA) compositions described herein are demonstrated to achieveprimary VE endpoints as discussed above, secondary VE endpoints (e.g.,confirmed severe COVID-19 in participants without evidence of infectionbefore vaccination and confirmed severe COVID-19 in all participants)can be evaluated sequentially, e.g., by the same method used for theprimary VE endpoint evaluation (hierarchical testing) as discussedabove. In some embodiments, evaluation of primary and/or secondary VEendpoints may be based on at least 20,000 or more subjects (e.g., atleast 25,000 or more subjects) randomized in a 1:1 ratio to the vaccineor placebo group, e.g., based on the following assumptions: (i) 1.0%illness rate per year in the placebo group, and (ii) 20% of theparticipants being non-evaluable or having serological evidence of priorinfection with SARS-CoV-2, potentially making them immune to furtherinfection.

In some embodiments, one or more RNA (e.g., mRNA) compositions describedherein may be administered according to a regimen established to achievemaintenance and/or continued enhancement of an immune response. Forexample, in some embodiments, an administration regimen may include afirst dose optionally followed by one or more subsequent doses; in someembodiments, need for, timing of, and/or magnitude of any suchsubsequent dose(s) may be selected to maintain, enhance, and/or modifyone or more immune responses or features thereof. In some embodiments,number, timing, and/or amount(s) of dose(s) have been established to beeffective when administered to a relevant population. In someembodiments, number, timing and/or amount(s) of dose(s) may be adjustedfor an individual subject; for example, in some embodiments, one or morefeatures of an immune response in an individual subject may be assessedat least once (and optionally more than once, for example multipletimes, typically spaced apart, often at pre-selected intervals) afterreceipt of a first dose. For example, presence of antibodies, B cells,and/or T cells (e.g., CD4⁺ and/or CD8⁺ T cells), and/or of cytokinessecreted thereby and/or identity of and/or extent of responses toparticular antigen(s) and/or epitope(s) may be assessed. In someembodiments, need for, timing of, and/or amount of a subsequent dose maybe determined in light of such assessments.

As noted hereinabove, in some embodiments, one or more subjects who havereceived an RNA (e.g., mRNA) composition as described herein may bemonitored (e.g., for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10days or more, including, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12 weeks or more, including for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 months or more,including for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years or more) fromreceipt of any particular dose to assess, for example, presence of animmune response to component(s) of the administered composition,evidence of exposure to and/or immune response to SARS-CoV-2 or anothercoronavirus, evidence of any adverse event, etc, including to performassessment of one or more of presence of antibodies, B cells, and/or Tcells (e.g., CD4⁺ and/or CD8⁺ T cells), and/or of cytokines secretedthereby and/or identity of and/or extent of responses to particularantigen(s) and/or epitope(s) may be assessed. Administration of acomposition as described herein may be in accordance with a regimen thatincludes one or more such monitoring steps.

For example, in some embodiments, need for, timing of, and/or amount ofa second dose relative to a first dose (and/or of a subsequent doserelative to a prior dose) is assessed, determined, and/or selected suchthat administration of such second (or subsequent) dose achievesamplification or modification of an immune response (e.g., as describedherein) observed after the first (or other prior) dose. In someembodiments, such amplification of an immune response (e.g., onesdescribed herein) may be at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, orhigher, as compared to the level of an immune response observed afterthe first dose. In some embodiments, such amplification of an immuneresponse may be at least 1.5 fold, at least 2-fold, at least 3-fold, atleast 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, atleast 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, atleast 30-fold, or higher, as compared to the level of an immune responseobserved after the first dose.

In some embodiments, need for, timing of, and/or amount of a second (orsubsequent) dose relative to a first (or other prior) dose is assessed,determined, and/or selected such that administration of the later doseextends the durability of an immune response (e.g., as described herein)observed after the earlier dose; in some such embodiments, thedurability may be extended by at least 1 week, at least 2 weeks, atleast 3 weeks, at least 1 month, at least 2 months, at least 3 months,at least 4 months, at least 5 months, at least 6 months, at least 7months, at least 8 months, at least 9 months, or longer. In someembodiments, an immune response observed after the first dose may becharacterized by production of neutralizing antibodies directed to aSARS-CoV-2 spike polypeptide and/or an immunogenic fragment thereof(e.g., RBD) as measured in serum from a subject and/or induction ofcell-mediated immune response (e.g., a T cell response againstSARS-CoV-2), including, e.g., in some embodiments induction of T cellsthat recognize at least one or more MHC-restricted (e.g., MHC classI-restricted) epitopes within a SARS-CoV-2 spike polypeptide and/or animmunogenic fragment thereof (e.g., RBD). In some embodiments, one ormore epitopes recognized by vaccine-induced T cells (e.g., CD8+ T cells)may be presented on a MHC class I allele that is present in at least 50%of subjects in a population, including, e.g., at least 60%, at least70%, at least 80%, at least 90%, or more; in some such embodiments, theMHC class I allele may be HLA-B*0702, HLA-A*2402, HLA-B*3501,HLA-B*4401, or HLA-A*0201. In some embodiments, an epitope may compriseHLA-A*0201 YLQPRTFLL (SEQ ID NO: 138); HLA-A*0201 RLQSLQTYV (SEQ ID NO:139); HLA-A*2402 QYIKWPWYI (SEQ ID NO: 140); HLA-A*2402 NYNYLYRLF (SEQID NO: 141); HLA-A*2402 KWPWYIWLGF (SEQ ID NO: 142); HLA-B*3501QPTESIVRF (SEQ ID NO: 143); HLA-B*3501 IPFAMQMAY (SEQ ID NO: 144); orHLA-B*3501 LPFNDGVYF (SEQ ID NO: 145).

In some embodiments, need for, timing of, and/or amount of a second doserelative to a first dose (or other subsequent dose relative to a priordose) is assessed, determined and/or selected such that administrationof such second (or subsequent) dose maintains or exceeds a referencelevel of an immune response; in some such embodiments, the referencelevel is determined based on human SARS-CoV-2 infection/COVID-19convalescent sera and/or PBMC samples drawn from subjects (e.g., atleast a period of time such as at least 14 days or longer, including,e.g., 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 25 days, 30days, 35 days, 40 days, 45 days, 50 days, 55 days, 60 days, or longer,after PCR-confirmed diagnosis when the subjects were asymptomatic. Insome embodiments, an immune response may be characterized by productionof neutralizing antibodies directed to a SARS-CoV-2 spike polypeptideand/or an immunogenic fragment thereof (e.g., RBD) as measured in serumfrom a subject and/or induction of cell-mediated immune response (e.g.,a T cell response against SARS-CoV-2), including, e.g., in someembodiments induction of T cells that recognize at least one or moreMHC-restricted (e.g., MHC class I-restricted) epitopes within aSARS-CoV-2 spike polypeptide and/or an immunogenic fragment thereof(e.g., RBD). In some embodiments, one or more epitopes recognized byvaccine-induced T cells (e.g., CD8+ T cells) may be presented on a MHCclass I allele that is present in at least 50% of subjects in apopulation, including, e.g., at least 60%, at least 70%, at least 80%,at least 90%, or more; in some such embodiments, the MHC class I allelemay be HLA-B*0702, HLA-A*2402, HLA-B*3501, HLA-B*4401, or HLA-A*0201. Insome embodiments, an epitope may comprise HLA-A*0201 YLQPRTFLL (SEQ IDNO: 138); HLA-A*0201 RLQSLQTYV (SEQ ID NO: 139); HLA-A*2402 QYIKWPWYI(SEQ ID NO: 140); HLA-A*2402 NYNYLYRLF (SEQ ID NO: 141); HLA-A*2402KWPWYIWLGF (SEQ ID NO: 142); HLA-B*3501 QPTESIVRF (SEQ ID NO: 143);HLA-B*3501 IPFAMQMAY (SEQ ID NO: 144); or HLA-B*3501 LPFNDGVYF (SEQ IDNO: 145).

In some embodiments, determination of need for, timing of, and/or amountof a second (or subsequent) dose may include one or more steps ofassessing, after (e.g., 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21 days or longer after) a first (or other prior) dose, presenceand/or expression levels of neutralizing antibodies directed to aSARS-CoV-2 spike polypeptide and/or an immunogenic fragment thereof(e.g., RBD) as measured in serum from a subject and/or induction ofcell-mediated immune response (e.g., a T cell response againstSARS-CoV-2), including, e.g., in some embodiments induction of T cellsthat recognize at least one or more MHC-restricted (e.g., MHC classI-restricted) epitopes within a SARS-CoV-2 spike polypeptide and/or animmunogenic fragment thereof (e.g., RBD). In some embodiments, one ormore epitopes recognized by vaccine-induced T cells (e.g., CD8+ T cells)may be presented on a MHC class I allele that is present in at least 50%of subjects in a population, including, e.g., at least 60%, at least70%, at least 80%, at least 90%, or more; in some such embodiments, theMHC class I allele may be HLA-B*0702, HLA-A*2402, HLA-B*3501,HLA-B*4401, or HLA-A*0201. In some embodiments, an epitope may compriseHLA-A*0201 YLQPRTFLL (SEQ ID NO: 138); HLA-A*0201 RLQSLQTYV (SEQ ID NO:139); HLA-A*2402 QYIKWPWYI (SEQ ID NO: 140); HLA-A*2402 NYNYLYRLF (SEQID NO: 141); HLA-A*2402 KWPWYIWLGF (SEQ ID NO: 142); HLA-B*3501QPTESIVRF (SEQ ID NO: 143); HLA-B*3501 IPFAMQMAY (SEQ ID NO: 144); orHLA-B*3501 LPFNDGVYF (SEQ ID NO: 145).

In some embodiments, a kit as provided herein may comprise a real-timemonitoring logging device, which, for example in some embodiments, iscapable of providing shipment temperatures, shipment time and/orlocation.

In some embodiments, an RNA (e.g., mRNA) composition as described hereinmay be shipped, stored, and/or utilized, in a container (such as a vialor syringe), e.g., a glass container (such as a glass vial or syringe),which, in some embodiments, may be a single-dose container or amulti-dose container (e.g., may be arranged and constructed to hold,and/or in some embodiments may hold, a single dose, or multiple doses ofa product for administration). In some embodiments, a multi-dosecontainer (such as a multi-dose vial or syringe) may be arranged andconstructed to hold, and/or may hold 2, 3, 4, 5, 6, 7, 8, 9, 10 or moredoses; in some particular embodiments, it may be designed to hold and/ormay hold 5 doses. In some embodiments, a single-dose or multi-dosecontainer (such as a single-dose or multi-dose vial or syringe) may bearranged and constructed to hold and/or may hold a volume or amountgreater than the indicated number of doses, e.g., in order to permitsome loss in transfer and/or administration. In some embodiments, an RNA(e.g., mRNA) composition as described herein may be shipped, stored,and/or utilized, in a preservative-free glass container (e.g., apreservative-free glass vial or syringe, e.g., a single-dose ormulti-dose preservative-free glass vial or syringe). In someembodiments, an RNA (e.g., mRNA) composition as described herein may beshipped, stored, and/or utilized, in a preservative-free glass container(e.g., a preservative-free glass vial or syringe, e.g., a single-dose ormulti-dose preservative-free glass vial or syringe) that contains afrozen liquid, e.g., in some embodiments 0.45 ml of frozen liquid (e.g.,including 5 doses). In some embodiments, an RNA (e.g., mRNA) compositionas described herein and/or a container (e.g., a vial or syringe) inwhich it is disposed, is shipped, stored, and/or utilized may bemaintained at a temperature below room temperature, at or below 4° C.,at or below 0° C., at or below −20° C., at or below −60° C., at or below−70° C., at or below −80° C., at or below −90° C., etc. In someembodiments, an RNA (e.g., mRNA) composition as described herein and/ora container (e.g., a viral or syringe) in which it is disposed, isshipped, stored, and/or utilized may be maintained at a temperaturebetween −80° C. and −60° C. and in some embodiments protected fromlight. In some embodiments, an RNA (e.g., mRNA) composition as describedherein and/or a container (e.g., a viral or syringe) in which it isdisposed, is shipped, stored, and/or utilized may be maintained at atemperature below about 25° C., and in some embodiments protected fromlight. In some embodiments, an RNA (e.g., mRNA) composition as describedherein and/or a container (e.g., a viral or syringe) in which it isdisposed, is shipped, stored, and/or utilized may be maintained at atemperature below about 5° C. (e.g., below about 4° C.), and in someembodiments protected from light. In some embodiments, an RNA (e.g.,mRNA) composition as described herein and/or a container (e.g., a viralor syringe) in which it is disposed, is shipped, stored, and/or utilizedmay be maintained at a temperature below about −20° C., and in someembodiments protected from light. In some embodiments, an RNA (e.g.,mRNA) composition as described herein and/or a container (e.g., a viralor syringe) in which it is disposed, is shipped, stored, and/or utilizedmay be maintained at a temperature above about −60° C. (e.g., in someembodiments at or above about −20° C., and in some embodiments at orabove about 4-5° C., in either case optionally below about 25° C.), andin some embodiments protected from light, or otherwise withoutaffirmative steps (e.g., cooling measures) taken to achieve a storagetemperature materially below about −20° C. In some embodiments, an RNA(e.g., mRNA) composition as described herein and/or a container (e.g., avial or syringe) in which it is disposed is shipped, stored, and/orutilized together with and/or in the context of a thermally protectivematerial or container and/or of a temperature adjusting material. Forexample, in some embodiments, an RNA (e.g., mRNA) composition asdescribed herein and/or a container (e.g., a vial or syringe) in whichit is disposed is shipped, stored, and/or utilized together with iceand/or dry ice and/or with an insulating material. In some particularembodiments, a container (e.g., a vial or syringe) in which an RNA(e.g., mRNA) composition is disposed is positioned in a tray or otherretaining device and is further contacted with (or otherwise in thepresence of) temperature adjusting (e.g., ice and/or dry ice) materialand/or insulating material. In some embodiments, multiple containers(e.g., multiple vials or syringes such as single use or multi-use vialsor syringes as described herein) in which a provided RNA (e.g., mRNA)composition is disposed are co-localized (e.g., in a common tray, rack,box, etc.) and packaged with (or otherwise in the presence of)temperature adjusting (e.g., ice and/or dry ice) material and/orinsulating material. To give but one example, in some embodiments,multiple containers (e.g., multiple vials or syringes such as single useor multi-use vials or syringes as described herein) in which an RNA(e.g., mRNA) composition is disposed are positioned in a common tray orrack, and multiple such trays or racks are stacked in a carton that issurrounded by a temperature adjusting material (e.g., dry ice) in athermal (e.g., insulated) shipper. In some embodiments, temperatureadjusting material is replenished periodically (e.g., within 24 hours ofarrival at a site, and/or every 2 hours, 4 hours, 6 hours, 8 hours, 10hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 1day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10days, etc.). Preferably, re-entry into a thermal shipper should beinfrequent, and desirably should not occur more than twice a day. Insome embodiments, a thermal shipper is re-closed within 5, 4, 3, 2, or 1minute, or less, of having been opened. In some embodiments, a providedRNA (e.g., mRNA) composition that has been stored within a thermalshipper for a period of time, optionally within a particular temperaturerange remains useful. For example, in some embodiments, if a thermalshipper as described herein containing a provided RNA (e.g., mRNA)composition is or has been maintained (e.g., stored) at a temperaturewithin a range of about 15° C. to about 25° C., the RNA (e.g., mRNA)composition may be used for up to 10 days; that is, in some embodiments,a provided RNA (e.g., mRNA) composition that has been maintained withina thermal shipper, which thermal shipper is at a temperature within arange of about 15° C. to about 25° C., for a period of not more than 10days is administered to a subject. Alternatively or additionally, insome embodiments, if a provided RNA (e.g., mRNA) composition is or hasbeen maintained (e.g., stored) within a thermal shipper, which thermalshipper has been maintained (e.g., stored) at a temperature within arange of about 15° C. to about 25° C., it may be used for up to 10 days;that is, in some embodiments, a provided RNA (e.g., mRNA) compositionthat has been maintained within a thermal shipper, which thermal shipperhas been maintained at a temperature within a range of about 15° C. toabout 25° C. for a period of not more than 10 days is administered to asubject.

In some embodiments, a provided RNA (e.g., mRNA) composition is shippedand/or stored in a frozen state. In some embodiments, a provided RNA(e.g., mRNA composition is shipped and/or stored as a frozen suspension,which in some embodiments does not contain preservative. In someembodiments, a frozen RNA (e.g., mRNA) composition is thawed. In someembodiments, a thawed RNA (e.g., mRNA) composition (e.g., a suspension)may contain white to off-white opaque amorphous particles. In someembodiments, a thawed RNA (e.g., mRNA) composition may be used for up toa small number (e.g., 1, 2, 3, 4, 5, or 6) of days after thawing ifmaintained (e.g., stored) at a temperature at or below room temperature(e.g., below about 30° C., 25° C., 20° C., 15° C., 10° C., 8° C., 4° C.,etc). In some embodiments, a thawed RNA (e.g., mRNA) composition may beused after being stored (e.g., for such small number of days) at atemperature between about 2° C. and about 8° C.; alternatively oradditionally, a thawed RNA (e.g., mRNA) composition may be used within asmall number (e.g., 1, 2, 3, 4, 5, 6) of hours after thawing at roomtemperature. Thus, in some embodiments, a provided RNA (e.g., mRNA)composition that has been thawed and maintained at a temperature at orbelow room temperature, and in some embodiments between about 2° C. andabout 8° C., for not more than 6, 5, 4, 3, 2, or 1 days is administeredto a subject. Alternatively or additionally, in some embodiments, aprovided RNA (e.g., mRNA) composition that has been thawed andmaintained at room temperature for not more than 6, 5, 4, 3, 2, or 1hours is administered to a subject. In some embodiments, a provided RNA(e.g., mRNA) composition is shipped and/or stored in a concentratedstate. In some embodiments, such a concentrated composition is dilutedprior to administration. In some embodiments, a diluted composition isadministered within a period of about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1hour(s) post-dilution; in some embodiments, such administration iswithin 6 hours post-dilution. Thus, in some embodiments, dilutedpreparation of a provided RNA (e.g., mRNA) composition is administeredto a subject within 6 hours post-dilution (e.g., as described hereinafter having been maintained at an appropriate temperature, e.g., at atemperature below room temperature, at or below 4° C., at or below 0°C., at or below −20° C., at or below −60° C., at or below −70° C., at orbelow −80° C., etc, and typically at or above about 2° C., for examplebetween about 2° C. and about 8° C. or between about 2° C. and about 25°C.). In some embodiments, unused composition is discarded within severalhours (e.g., about 10, about 9, about 8, about 7, about 6, about 5 orfewer hours) after dilution; in some embodiments, unused composition isdiscarded within 6 hours of dilution.

In some embodiments, an RNA (e.g., mRNA) composition that is stored,shipped or utilized (e.g., a frozen composition, a liquid concentratedcomposition, a diluted liquid composition, etc.) may have beenmaintained at a temperature materially above −60° C. for a period oftime of at least 1, 2, 3, 4, 5, 6, 7 days or more, or at least 1, 2, 3,4, 5, 6, 7, 8, 9, 10 weeks or more, or at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12 months or more; in some such embodiments, such compositionmay have been maintained at a temperature at or above about −20° C. forsuch period of time, and/or at a temperature up to or about 4-5° C. forsuch period of time, and/or may have been maintained at a temperatureabove about 4-5° C., and optionally about 25° C. for a period of time upthat is less than two (2) months and/or optionally up to about one (1)month. In some embodiments, such composition may not have been stored,shipped or utilized (or otherwise exposed to) a temperature materiallyabove about 4-5° C., and in particular not at or near a temperature ofabout 25° C. for a period of time as long as about 2 weeks, or in someembodiments 1 week. In some embodiments, such composition may not havebeen stored, shipped or utilized (or otherwise exposed to) a temperaturematerially above about −20° C., and in particular not at or near atemperature of about 4-5° C. for a period of time as long as about 12months, 11 months, 10 months, 9 months, 8 months, 7 months, 6 months, 5months, 4 months, 3 months, 2 months, or, in some embodiments, for aperiod of time as long as about 8 weeks or 6 weeks or materially morethan about 2 months or, in some embodiments, 3 months or, in someembodiments 4 months.

In some embodiments, an RNA (e.g., mRNA) composition that is stored,shipped or utilized (e.g., a frozen composition, a liquid concentratedcomposition, a diluted liquid composition, etc.) may be protected fromlight. In some embodiments, one or more steps may be taken to reduce orminimize exposure to light for such compositions (e.g., which may bedisposed within a container such as a vial or a syringe). In someembodiments, exposure to direct sunlight and/or to ultraviolent light isavoided. In some embodiments, a diluted solution may be handled and/orutilized under normal room light conditions (e.g., without particularsteps taken to minimize or reduce exposure to room light). It should beunderstood that strict adherence to aseptic techniques is desirableduring handling (e.g., diluting and/or administration) of an RNA (e.g.,mRNA) composition as described herein. In some embodiments, an RNA(e.g., mRNA) composition as described herein is not administered (e.g.,is not injected) intravenously. In some embodiments, an RNA (e.g., mRNA)composition as described herein is not administered (e.g., is notinjected) intradermally. In some embodiments, an RNA (e.g., mRNA)composition as described herein is not administered (e.g., is notinjected) subcutaneously. In some embodiments, an RNA (e.g., mRNA)composition as described herein is not administered (e.g., is notinjected) any of intravenously, intradermally, or subcutaneously. Insome embodiments, an RNA (e.g., mRNA) composition as described herein isnot administered to a subject with a known hypersensitivity to anyingredient thereof. In some embodiments, a subject to whom an RNA (e.g.,mRNA) composition has been administered is monitored for one or moresigns of anaphylaxis. In some embodiments, a subject to whom an RNA(e.g., mRNA) composition is administered had previously received atleast one dose of a different vaccine for SARS-CoV-2; in someembodiments, a subject to whom an RNA (e.g., mRNA) composition isadministered had not previously received a different vaccine forSARS-CoV-2. In some embodiments, a subject's temperature is takenpromptly prior to administration of an RNA (e.g., mRNA) composition(e.g., shortly before or after thawing, dilution, and/or administrationof such composition); in some embodiments, if such subject is determinedto be febrile, administration is delayed or canceled. In someembodiments, an RNA (e.g., mRNA) composition as described herein is notadministered to a subject who is receiving anticoagulant therapy or issuffering from or susceptible to a bleeding disorder or condition thatwould contraindicate intramuscular injection. In some embodiments, anRNA (e.g., mRNA) composition as described herein is administered by ahealthcare professional who has communicated with the subject receivingthe composition information relating to side effects and risks. In someembodiments, an RNA (e.g., mRNA) composition as described herein isadministered by a healthcare professional who has agreed to submit anadverse event report for any serious adverse events, which may includefor example one or more of death, development of a disability orcongenital anomaly/birth defect (e.g., in a child of the subject),in-patient hospitalization (including prolongation of an existinghospitalization), a life-threatening event, a medical or surgicalintervention to prevent death, a persistent or significant orsubstantial disruption of the ability to conduct normal life functions;or another important medical event that may jeopardize the individualand may require medical or surgical intervention (treatment) to preventone of the other outcomes. In some embodiments, provided RNAcompositions are administered to a population of individuals under 18years of age, or under 17 years of age, or under 16 years of age, orunder years of age, or under 14 years of age, or under 13 years of age,for example according to a regimen established to have a rate ofincidence for one or more of the local reaction events indicated belowthat does not exceed the rate of incidence indicated below:

-   -   pain at the injection site (75% after a first dose and/or a        second dose, and/or a lower incidence after a second dose, e.g.,        65% after a second dose);    -   redness at the injection site (less than 5% after a first dose        and/or a second dose); and/or    -   swelling at the injection site (less than 5% after a first dose        and/or a second dose).

In some embodiments, provided RNA compositions are administered to apopulation of individuals under 18 years of age, or under 17 years ofage, or under 16 years of age, or under years of age, or under 14 yearsof age, or under 13 years of age, for example according to a regimenestablished to have a rate of incidence for one or more of the systemicreaction events indicated below that does not exceed the rate ofincidence indicated below:

-   -   fatigue (55% after a first dose and/or a second dose);    -   headache (50% after a first dose and/or a second dose);    -   muscle pain (40% after a first dose and/or a second dose);    -   chills (40% after a first dose and/or a second dose);    -   joint pain (20% after a first dose and/or a second dose);    -   fever (25% after a first dose and/or a second dose);    -   vomiting (10% after a first dose and/or a second dose); and/or    -   diarrhea (10% after a first dose and/or a second dose).

In some embodiments, medication that alleviates one or more symptoms ofone or more local reaction and/or systemic reaction events (e.g.,described herein) are administered to individuals under 18 years of age,or under 17 years of age, or under 16 years of age, or under years ofage, or under 14 years of age, or under 13 years of age who have beenadministered with provided RNA compositions and have experienced one ormore of the local and/or systemic reaction events (e.g., describedherein). In some embodiments, antipyretic and/or pain medication can beadministered to such individuals.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 . Schematic overview of the S protein organization of theSARS-CoV-2 S protein.

The sequence within the S1 subunit consists of the signal sequence (SS)and the receptor binding domain (RBD) which is the key subunit withinthe S protein which is relevant for binding to the human cellularreceptor ACE2. The S2 subunit contains the S2 protease cleavage site(S2′) followed by a fusion peptide (FP) for membrane fusion, heptadrepeats (HR1 and HR2) with a central helix (CH) domain, thetransmembrane domain (TM) and a cytoplasmic tail (CT).

FIG. 2 . Exemplary SARS-CoV-2 vaccine constructs.

Based on the full and wildtype S protein, we have designed differentconstructs encoding the (1) full protein with mutations in closedistance to the first heptad repeat (HRP1) that include stabilizingmutations preserving neutralisation sensitive sites, the (2) S1 domainor the (3) RB domain (RBD) only. Furthermore, to stabilize the proteinfragments a fibritin domain (F) was fused to the C-terminus. Allconstructs start with the signal peptide (SP) to ensure Golgi transportto the cell membrane.

FIG. 3 . General structure of the RNA.

Schematic illustration of the general structure of the RNA vaccines with5′-cap, 5′- and 3′-untranslated regions, coding sequences with intrinsicsecretory signal peptide as well as GS-linker, and poly(A)-tail. Pleasenote that the individual elements are not drawn exactly true to scalecompared to their respective sequence lengths.

UTR=Untranslated region; sec=Secretory signal peptide; RBD=ReceptorBinding Domain; GS=Glycine-serine linker.

FIG. 4 . General structure of the RNA.

Schematic illustration of the general structure of RNA drug substanceswith 5′-cap, 5′- and 3′-untranslated regions, coding sequences withintrinsic secretory signal peptide as well as GS-linker, andpoly(A)-tail. Please note that the individual elements are not drawnexactly true to scale compared to their respective sequence lengths.

GS=Glycine-serine linker; UTR=Untranslated region; Sec=Secretory signalpeptide; RBD=Receptor Binding Domain.

FIG. 5 . General structure of the RNA.

Schematic illustration of the general structure of RNA vaccines with5′-cap, 5′- and 3′-untranslated regions, coding sequences of theVenezuelan equine encephalitis virus (VEEV) RNA-dependent RNA polymerasereplicase and the SARS-CoV-2 antigen with intrinsic secretory signalpeptide as well as GS-linker, and poly(A)-tail. Please note that theindividual elements are not drawn exactly true to scale compared totheir respective sequence lengths.

UTR=Untranslated region; Sec=Secretory signal peptide; RBD=ReceptorBinding Domain; GS=Glycine-serine linker.

FIG. 6 . Schematic overview of the S protein organization of theSARS-CoV-2 S protein and constructs for the development of a SARS-CoV-2vaccine.

Based on the wildtype S protein, we have designed two differenttransmembrane-anchored RBD-based vaccine constructs encoding the RBDfragment fused to the T4 fibritin trimerization domain (F) and theautochthonus transmembrane domain (TM). Construct (1) starts with theSARS-CoV-2-S signal peptide (SP; AA 1-19 of the S protein) whereasconstruct (2) starts with the human Ig heavy chain signal peptide(huSec) to ensure Golgi transport to the cell membrane.

FIG. 7 . Anti-S protein IgG response 6, 14 and 21 d after immunizationwith LNP-C12 formulated modRNA coding for transmembrane-anchoredRBD-based vaccine constructs.

BALB/c mice were immunized IM once with 4 μg of LNP-C12-formulatedtransmembrane-anchored RBD-based vaccine constructs (surrogate toBNT162b3c/BNT162b3d). On day 6, 14 and 21 after immunization, animalswere bled and the serum samples were analyzed for total amount ofanti-S1 (left) and anti-RBD (right) antigen specific immunoglobulin G(IgG) measured via ELISA. For day 6 (1:50), day 14 (1:300) and day 21(1:900) different serum dilution were included in the graph. One pointin the graph stands for one mouse, every mouse sample was measured induplicates (group size n=8; mean+SEM is included for the groups).

FIG. 8 . Neutralization of SARS-CoV-2 pseudovirus 6, 14 and 21 d afterimmunization with LNP-C12 formulated modRNA coding fortransmembrane-anchored RBD-based vaccine constructs.

BALB/c mice were immunized IM once with 4 μg of LNP-C12-formulatedtransmembrane-anchored RBD-based vaccine constructs (surrogate toBNT162b3c/BNT162b3d). On day 6, 14 and 21 after immunization, animalswere bled and the sera were tested for SARS CoV-2 pseudovirusneutralization. Graphs depict pVN₅₀ serum dilutions (50% reduction ofinfectious events, compared to positive controls without serum). Onepoint in the graphs stands for one mouse. Every mouse sample wasmeasured in duplicate. Group size n=8. Mean+SEM is shown by horizontalbars with whiskers for each group. LLOQ lower limit of quantification.ULOQ, upper limit of quantification.

FIG. 9 . 50% pseudovirus neutralization titers (pVNT₅₀) of seracollected 21 days after the second dose and 1 month after the third doseof BNT162b2 against VSV-SARS-CoV-2-S pseudovirus bearing the Wuhan Hu-1reference or Omicron lineage spike protein. N=19-20 sera from immunizedsubjects collected either 21 days after the second BNT162b2 dose or 1months after the third BNT162b2 dose were tested. For values below thelimit of detection (LOD; 10), LOD/2 values are plotted. Group GMTs(values above bars) with 95% confidence intervals are shown.

FIG. 10 . CD8+ T cell epitopes in BNT162b2 vaccine remain largelyunaffected by Omicron BA.1 variant mutations. Shown is the number ofpreviously identified MHC-I epitopes affected in various variants ofconcern (VOCs). Approximately 80% of previously identified CD8+ epitopesare not affected by the mutations in the BA.1 Omicron variant,suggesting that two doses of BNT162b2 may still induce protectionagainst severe disease.

FIG. 11 . Neutralization of Omicron BA.1 after two doses of BNT162b2 andvariant specific booster. Shown is neutralization of the Omicron BA.1variant from sera of patients administered two doses of BNT162b2 and (i)a third booster dose of BNT162b2, or (ii) a third booster dose of an RNAencoding a Spike protein with alpha or delta variant mutations, or athird booster dose of both a Spike protein comprising alpha mutationsand a Spike protein comprising delta mutations. The values are derivedfrom separate neutralization GMTs from the pseudovirus testing. Alsoshown is a schematic depicting a process for developing new SARS-CoV-2variant specific vaccines.

FIG. 12 . Longitudinal analysis of neutralizing antibody responsesagainst VSV-SARS-CoV-2-S pseudovirus bearing the Wuhan or Omicron BA.1variant spike protein in a subset of study participants. Sera from n=9participants drawn at 21 days after dose 2, prior to dose 3, 1 monthafter dose 3 and 3 months after dose 3 were tested. Each serum wastested in duplicate and individual geometric mean 50% pseudovirusneutralizing titers (GMTs) were calculated. For values below the limitof detection (LOD), LOD/2 values were assigned. Group GMTs (values intable) and 95% confidence intervals per timepoint are indicated.

FIG. 13 . Analysis of HLA class I T cell epitopes conservation betweenthe Wuhan and Omicron BA.1 variants. HLA class I restricted Spikeprotein epitopes with T cell reactivity identified based on theirrecognition by CDB+ T cells and reported in IEDB (n=244) are plotted bytheir position (top row) along the Spike protein (bottom row). Epitopeindications are positioned by the amino acid position of the center ofthe epitope; epitopes conserved in both variants are marked in lightgrey (n=208); while epitopes spanning an Omicron BA.1 mutation site aremarked dark grey (n=36). NTD=N-terminal domain; RBD=Receptor-bindingdomain; FCS=Furin cleavage site. The S1 and S2 regions of the Spikeprotein are indicated.

FIG. 14 . Schematics of an exemplary vaccination regimen.

FIG. 15 . Cohorts, sampling and experimental setup for characterizationof immune response in Omicron breakthrough cases. Blood samples weredrawn from four cohorts: Omicron-naïve individuals double- ortriple-vaccinated with BNT162b2, and individuals double- ortriple-vaccinated with BNT162b2 that subsequently had a breakthroughinfection with Omicron BA.1. PBMCs and sera were isolated in theOmicron-naïve cohorts at the timepoints indicated following their mostrecent vaccination; for convalescent cohorts, the time from their mostrecent vaccination to Omicron BA.1 infection, and infection to PBMC andserum isolation are indicated (all values specified as median-range).Serum neutralizing capacity was assessed using a pseudovirus and livevirus neutralization test; SARS-CoV-2 spike-specific B_(MEM) cells wereassessed via a flow cytometry-based B cell phenotyping assay using bulkPBMCs. N/A, not applicable.

FIG. 16 . Omicron breakthrough infection in BNT162b2 double- andtriple-vaccinated individuals induces broad neutralization of OmicronBA.1, BA.2 and other VOCs.

Serum was drawn from double-vaccinated individuals (BNT162b2²) at 22days after the second dose (open circles), from triple-vaccinatedindividuals (BNT162b2³) at 28 days after the third dose (closedcircles), from double-vaccinated individuals with an Omicronbreakthrough infection (BNT162b2²+Omi) at 46 days post-infection (opentriangles), and from triple-vaccinated individuals and Omicronbreakthrough infection (BNT162b2³+Omi) at 44 days post-infection (closedtriangles). Serum was tested in duplicate; (A) shows 50% pseudovirusneutralization (pVN₅₀) geometric mean titers (GMTs), (B) shows 50% virusneutralization (VN₅₀) GMTs, and (C) shows the geometric mean ratio ofSARS-CoV-2 variant of concern (VOC) and Wuhan VN₅₀ GMTs. For titervalues below the limit of detection (LOD), LOD/2 values were plotted.Values above violin plots represent group GMTs. The non-parametricFriedman test with Dunn's multiple comparisons correction was used tocompare Wuhan neutralizing group GMTs with titers against the indicatedvariants and SARS-CoV-1. Multiplicity-adjusted p values are shown. (A)pVN₅₀ GMTs against Wuhan, VOC and SARS-CoV-1 pseudovirus. (B) VN₅₀ GMTsagainst live SARS-CoV-2 Wuhan, Beta, Delta and Omicron BA.1. (C) Groupgeometric mean ratios with 95% confidence intervals for all cohortsshown in (B).

FIG. 17 . B_(MEM) cells of individuals double- and triple-vaccinatedwith BNT162b2 broadly recognize VOCs and are further boosted by Omicronbreakthrough infection. PBMC samples from double-vaccinated individuals(BNT162b2²) at 22 days after the second dose (open squares) and 5 monthsafter the second dose (open circles), from triple-vaccinated individuals(BNT162b2³) at 84 days after the third dose (closed circles), fromdouble-vaccinated individuals with Omicron breakthrough infection(BNT162b2²+Omi) at 46 days post-infection (open triangles), and fromtriple-vaccinated individuals with Omicron breakthrough infection(BNT162b23+Omi) at 44 days post-infection (closed triangles) wereanalyzed via flow cytometry for SARS-CoV-2-specific B_(MEM) cell(B_(MEM)—CD3-CD19+CD20+IgD-CD38^(int/low)) frequencies via B cell baitstaining. (A) Schematic of one-dimensional staining of B_(MEM) cellswith fluorochrome-labeled SARS-CoV-2 S protein tetramer bait allowingdiscrimination of variant recognition. Frequencies of Wuhan or VOCfull-length S protein-(B) and RBD-(C) specific B_(MEM) cells for thefour groups of individuals were analyzed. Variant-specific B_(MEM) cellfrequencies were normalized to Wuhan frequencies for S protein (D) andRBD-(E) binding. (F) Depicts the frequency ratios of RBD proteinspecific B_(MEM) cells over full-length S protein-specific B_(MEM)cells.

FIG. 18 . Omicron breakthrough infection of BNT162b2 double- andtriple-vaccinated individuals primarily boosts B_(MEM) against conservedepitopes shared broadly between S proteins of Wuhan and other VOCsrather than strictly Omicron S-specific epitopes. PBMC samples fromdouble-vaccinated individuals (BNT162b2²) at 22 days after the seconddose (open squares) and 5 months after the second dose (open circles),from triple-vaccinated individuals (BNT162b2³) at 84 days after thethird dose (closed circles), from double-vaccinated individuals withOmicron breakthrough infection (BNT162b2²+Omi) at 46 days post-infection(open triangles), and from triple-vaccinated individuals with Omicronbreakthrough infection (BNT162b2³+Omi) at 44 days post-infection (closedtriangle) were analyzed via flow cytometry for SARS-CoV-2-specificmemory B cell (B_(MEM)—CD3-CD19+CD20+IgD-CD38^(int/low)) frequencies viaB cell bait staining (schematic shown in (A)). (B) shows representativeflow plots of Omicron and Wuhan S protein- and RBD-binding for each ofthe four groups of individuals investigated. Frequencies of B_(MEM)binding Omicron, Wuhan, or both (shared) shown for full-length S proteinin (C) and RBD shown in (D) for Omicron-experienced and naïve BNT162b2double and triple vaccines. (E) Venn diagrams visualizing thecombinatorial (Boolean) gating strategy to identify cross-reactiveB_(MEM) recognizing all four variants simultaneously (All 4+ve) andB_(MEM) recognizing only Omicron (only Omi) or only Wuhan (only Wuhan) Sproteins. Frequencies for these three B_(MEM) sub-groups were comparedfor full-length S protein (F) and RBD (G) in the four different groupsof individuals investigated. RBD variant recognition pattern by B_(MEM)was assessed through Boolean flow cytometric gating strategy andfrequencies recognizing combinations of Wuhan and/or variant RBDs (SEQID NO: 124-128) are displayed in (H), for all Omicron convalescentsubjects (double and triple vaccines pooled, n=13). (I) Conserved sitewithin the RBD domain recognized by RBD-specific B_(MEM) after Omicronbreak-through infection. Mean values are indicated in C, D, F, and G.n=number of individuals per group.

FIG. 19 . Omicron breakthrough infection of individuals vaccinated withother approved COVID-19 vaccines or mixed regimens results in immunesera that broadly neutralize Omicron BA.1, BA.2 and other VOCs plusSARS-CoV-1. Serum was drawn from 10 individuals vaccinated with otherapproved COVID-19 vaccines or mixed regimens at a median of 43 daysafter infection (diamonds). Serum was tested in duplicate; individual50% pseudovirus neutralization (pVN₅₀) geometric mean titers (GMTs)against SARS-CoV-2 Wuhan, Alpha, Beta, Delta and Omicron BA.1 and BA.2variants, plus SARS-CoV-1 were plotted. For titer values below the limitof detection (LOD), LOD/2 values were plotted. Values above violin plotsrepresent group GMTs. The non-parametric Friedman test with Dunn'smultiple comparisons correction was used to compare Wuhan neutralizinggroup GMTs with titers against the indicated variants and SARS-CoV-1.Multiplicity-adjusted p values are shown. Approved vaccines includedAZD1222, BNT162b2 (in some embodiments as part of a 4-dose series),Ad26.COV2.S, mRNA-1273 (administered as a two-dose or three-doseseries), and combinations thereof.

FIG. 20 . 50% neutralization titers of sera collected 1 month after afourth dose of BNT162b2 or an Omicron BA.1-specific booster. Subjectswho were previously administered two doses of BNT162b2, and a third(booster) dose of BNT162b2 (30 ug) received a dose (30 ug) of (i) an RNAencoding a SARS-CoV-2 S protein from an Omicron BA.1 variant (e.g., asdescribed herein (referred to herein as “Omicron BA.1-specific RNAvaccine”), or (ii) BNT162b2, as a fourth (booster) dose. Serum from thesubjects were collected one month after administration of the 4th(booster) dose. Group GMTs (values above bars) with 95% confidenceintervals are shown. “b2” refers to sera from subjects administeredWuhan-specific RNA vaccine as the 4^(th) (booster) dose of BNT162b2.“OMI” refers to sera from subjects administered an Omicron BA.1-specific4th (booster) dose. Also shown is the fold-change in titer from beforeadministration of the 4^(th) dose to after administration of the 4thdose (Pre/Post Vax Fold-Rise), and the ratio of geometric mean ratio(GMR) and geometric mean fold rise (GMFR) observed in subjectsadministered a 4^(th) dose of an Omicron BA.1-specific RNA vaccine asthe 4^(th) dose, as compared to subjects administered BNT162b2 as the4^(th) dose. “FFRNT” refers to fluorescent focus reductionneutralization test. Neutralization data was obtained using an FFRNTassay, with a viral particle containing a SARS-CoV-2 S protein from thevariant indicated in the figures. (A) Comparison of titers ofneutralizing antibodies against a SARS-CoV-2-S pseudovirus comprising aSARS-CoV-2 S protein having mutations characteristics of an Omicron BA.1variant. Sera from subjects previously or currently infected withSARS-CoV-2 excluded. (B) Comparison of titers of neutralizing antibodiesagainst a SARS-CoV-2 pseudovirus comprising a SARS-CoV-2 S proteinhaving mutations characteristics of an Omicron BA.1 variant in sera froma population that includes subjects previously or currently infectedwith SARS-CoV-2 (as determined by an antigen assay or a PCR assayrespectively). (C) Comparison of titers of neutralizing antibodiesagainst a SARS-CoV-2 pseudovirus comprising a SARS-CoV-2 S protein froma Wuhan strain. Sera from subjects previously or currently infected withSARS-CoV-2 excluded. (D) Comparison of titers of neutralizing antibodiesagainst a SARS-CoV-2 pseudovirus comprising a SARS-CoV-2 S protein froma Wuhan strain, in sera from a population comprising individualspreviously or currently infected with SARS-CoV-2 (as determined by anantigen assay or a PCR assay, respectively. (E) Comparison of titers ofneutralizing antibodies against a SARS-CoV-2 pseudovirus comprising aSARS-CoV-2 S protein having mutations characteristics of a deltavariant. Sera from subjects previously or currently infected withSARS-CoV-2 excluded. (F) Comparison of titers of neutralizationantibodies against a SARS-CoV-2 pseudovirus comprising a SARS-CoV-2protein having mutations characteristic of a delta variant, in sera froma population including subjects previously or currently infected withSARS-CoV-2 (as determined by an antigen assay or a PCR assay,respectively).

FIG. 21 . Neutralization of SARS-CoV-2 pseudovirus 7 days afterimmunization with modRNA coding for variant specific S proteins. Micewere immunized twice with LNP-formulated vaccine comprising (i) BNT162b2(encoding a SARS-CoV-2 S protein from a Wuhan strain), (ii) RNA encodinga SARS-CoV-2 S protein having mutations characteristic of an OmicronBA.1 variant (Omi), (iii) RNA encoding an S protein having mutationscharacteristic of a delta variant, (iv) a combination of BNT162b2 and anRNA encoding an protein having mutations characteristic of an OmicronBA.1 variant (B2+Omi), or (v) RNA encoding a SARS-CoV-2 S protein havingmutations characteristic of a delta variant and RNA encoding aSARS-CoV-2 S protein having mutations characteristic of an Omicron BA.1variant (Delta+Omi). 7 days after the second immunization, animals werebled and sera was tested for neutralization of a SARS-CoV-2-Spseudovirus comprising a SARS-CoV-2 S protein from a Wuhan strain, or aSARS-CoV-2 S protein having mutations characteristic of a beta, delta,or Omicron BA.1 variant. Graphs depict pVN₅₀ serum dilutions (50%reduction of infectious events, compared to positive controls withoutserum). One point in the graphs stands for one mouse. Every mouse samplewas measured in duplicate. Mean+SEM is shown by horizontal bars withwhiskers for each group. LLOD, lower limit of detection. ULOD, upperlimit of detection.

FIG. 22 . RNA encoding a SARS-CoV-2 S protein having mutationscharacteristic of a Beta variant increases neutralization antibodytiters against SARS-CoV-2 when administered to patients previouslyadministered two doses of a vaccine encoding a SARS-CoV-2 S protein of aWuhan strain. Subjects previously administered two doses of an RNAvaccine encoding a SARS-CoV-2 S protein of a Wuhan strain wereadministered a third and a fourth dose of an RNA vaccine encoding aSARS-CoV-2 S protein having mutations characteristic of a Beta variant.Neutralization antibody titers were measured before administration of anRNA vaccine encoding a SARS-CoV-2 S protein of a Wuhan strain(D1-PreVax), one month after administration of a second dose of an RNAvaccine encoding a SARS-CoV-2 S protein of a Wuhan strain (M1PD2),one-month after administration of a third dose of an RNA vaccineencoding a SARS-CoV-2 S protein having mutations characteristic of aSARS-CoV-2 Beta variant, and one month after administration of a fourthdose of an RNA vaccine encoding a SARS-CoV-2 S protein having mutationscharacteristic of a SARS-CoV-2 Beta variant. The third and fourth doseswere administered 1 month apart from one another. GMFR refers to thegeometric mean fold rise, and is a measure of the increase inneutralization antibody titers since the previous vaccine dose (e.g.,the GMFR for Post-Dose2 (PD2) is a measure of the increase inneutralization antibody titers relative to before administration of anyvaccine (pre-vax)). (A) Neutralization antibody titers measured in aviral neutralization assay that uses a viral particle comprising aSARS-CoV-2 S protein of a Wuhan strain. (B) Neutralization antibodytiters measured in a viral neutralization assay that uses a viralparticle comprising a SARS-CoV-2 S protein having mutationscharacteristic of a Beta variant.

FIG. 23 . 50% neutralization titers of sera collected 7 days after afourth dose of BNT162b2, an Omicron BA.1-specific booster, or a bivalentvaccine. Subjects who were previously administered two doses of BNT162b2(30 ug), and a third (booster) dose of BNT162b2 (30 ug) received (i) a30 ug dose of BNT162b2 (encoding a SARS-CoV-2 S protein from a Wuhanstrain), (ii) a 60 ug dose of BNT162b2, (iii) a 30 ug dose of RNAencoding a SARS-CoV-2 S protein having mutations characteristic of anOmicron BA.1 variant (e.g., as described herein (referred to herein as“Omicron BA.1-specific RNA vaccine”)), (iii) a 60 ug dose of RNAencoding a SARS-CoV-2 S protein having mutations characteristic of anOmicron BA.1 variant, (iv) a 30 ug dose of a bivalent vaccine,comprising 15 ug of BNT162b2 and 15 ug of RNA encoding a SARS-CoV-2 Sprotein comprising mutations characteristic of an Omicron BA.1 variant,or (v) a 60 ug dose of a bivalent vaccine, comprising 30 ug of BNT162b2and 30 ug of RNA encoding a SARS-CoV-2 S protein comprising mutationscharacteristic of an Omicron BA.1 variant. Geometric mean ratio (GMR) oftiters in serum from subjects were collected 7 days after administrationof a 4th dose. “b2” refers to sera from subjects administered aWuhan-specific RNA vaccine as a 4^(th) dose of BNT162b2. “OMI” refers tosera from subjects administered an Omicron BA.1-specific 4^(th) dose.“Bivalent” refers to sera from subjects administered a compositioncomprising BNT162b2 and an RNA encoding a SARS-CoV-2 S proteincomprising mutations that are characteristic of an Omicron BA.1 variantas a 4^(th) dose. Also shown is the fold-rise in titer from beforeadministration of a 4^(th) dose to 7 days after administration of a4^(th) dose (*Fold-Rise). “FFRNT” refers to fluorescent focus reductionneutralization test. Neutralization data was obtained using an FFRNTassay, with a viral particle containing a SARS-CoV-2 S protein havingmutations characteristic of the variant indicated in the figures. LLOQrefers to Lower Limit of Quantification and ULOQ refers to Upper Limitof Quantification. (A) Comparison of titers of neutralizing antibodiesagainst a SARS-CoV-2 pseudovirus comprising a SARS-CoV-2 S proteinhaving mutations characteristics of an Omicron BA.1 variant. Sera fromsubjects previously or currently infected with SARS-CoV-2 excluded. (B)Comparison of titers of neutralizing antibodies against a SARS-CoV-2pseudovirus comprising a SARS-CoV-2 S protein having mutationscharacteristics of an Omicron BA.1 variant in sera from a populationthat includes subjects previously or currently infected with SARS-CoV-2(e.g., as determined by an antibody test or a PCR assay respectively).(C) Comparison of titers of neutralizing antibodies against a SARS-CoV-2pseudovirus comprising a SARS-CoV-2 S protein of a Wuhan strain. Serafrom subjects previously or currently infected with SARS-CoV-2 excluded.(D) Comparison of titers of neutralizing antibodies against a SARS-CoV-2pseudovirus comprising a SARS-CoV-2 S protein of a Wuhan strain, in serafrom a population that includes individuals previously or currentlyinfected with SARS-CoV-2. (E) Comparison of titers of neutralizingantibodies against a SARS-CoV-2 pseudovirus comprising a SARS-CoV-2 Sprotein having mutations characteristics of a Delta variant. Sera fromsubjects previously or currently infected with SARS-CoV-2 excluded. (F)Comparison of titers of neutralization antibodies against a SARS-CoV-2pseudovirus comprising a SARS-CoV-2 S protein having mutationscharacteristic of a Delta variant, in sera from a population includingsubjects previously or currently infected with SARS-CoV-2. (G) Geometricmean rise (GMR) of neutralization antibodies observed in subjectsadministered 60 ug of BNT162b2, 30 ug of RNA encoding a SARS-CoV-2 Sprotein having mutations characteristic of an Omicron BA.1 variant (OMI30 ug), 60 ug of RNA encoding a SARS-CoV-2 S protein having mutationscharacteristic of an Omicron BA.1 variant (OMI 60 ug), ug of a bivalentvaccine comprising 15 ug of BNT162b2 and 15 ug of RNA encoding aSARS-CoV-2 S protein having mutations characteristic of an Omicron BA.1variant (Bivalent 30 ug), or 60 ug of a bivalent vaccine comprising 30ug of BNT162b2 and 30 ug of RNA encoding a SARS-CoV-2 S protein havingmutations characteristic of an Omicron BA.1 variant (Bivalent 60 ug), ascompared to subjects administered 30 ug of BNT162b2 as a 4^(th) dose.Results are shown both for a population pool that excludes subjectspreviously or currently infected with SARS-CoV-2 and a population poolthat includes these subjects.

FIG. 24 . Reactogenicity of certain exemplary RNA (formulated in LNP) ata given dose: subjects administered a 60 ug dose of RNA encoding aSARS-CoV-2 S protein are more likely to exhibit a higher injection sitepain and exhibit similar systemic reactions as subjects administered a30 ug dose of RNA. Subjects were administered 30 ug or 60 ug of RNAencoding a SARS-CoV-2 S protein from a Wuhan strain (BNT162b2,corresponding to groups G1 and G2, respectively), 30 ug or 60 ug of RNAencoding a SARS-CoV-2 S protein having mutations characteristic of anOmicron BA.1 variant (BNT162b2 OMI, corresponding to groups G3 and G4,respectively), 30 ug of a bivalent vaccine comprising 15 ug of RNAencoding a SARS-CoV-2 S protein from a Wuhan strain and 15 ug of RNAencoding a SARS-CoV-2 S protein having mutations characteristic of anOmicron BA.1 variant (BNT162b2 (15 ug)+BNT162b2 OMI (15 ug),corresponding to group G5), or 60 ug of a bivalent vaccine comprising 30ug of RNA encoding a SARS-CoV-2 S protein from a Wuhan strain and 30 ugof RNA encoding a SARS-CoV-2 S protein having mutations characteristicof an Omicron BA.1 variant (BNT162b2 (30 ug)+BNT162b2 OMI (30 ug),corresponding to group G6). (A) Local reactions, including redness,swelling, and pain at the injection site, observed within 7 days ofinjection. Injection site pain was found to be increased in subjectsadministered 60 ug of RNA encoding a SARS-CoV-2 S protein comprisingmutations characteristic an Omicron BA.1 variant or a bivalent vaccine,as compared to other doses tested. (B) Systemic reactions, includingfever, fatigue, headache, chills, vomiting, diarrhea, muscle pain, jointpain, and use of medication, observed within 7 days of injection.Systemic reactions through 7 days were observed to be broadly similaracross different groups. Fatigue was found to trend higher afteradministration of 60 ug doses, as compared to 30 ug doses.

FIG. 25 . 50% neutralization titers of sera collected 1 month after afourth dose of BNT162b2, an Omicron BA.1-specific booster, or a bivalentvaccine against an Omicron BA.1 variant. Subjects who were previouslyadministered two doses of BNT162b2 (30 ug), and a third (booster) doseof BNT162b2 (30 ug) were administered (i) a 30 ug dose of BNT162b2(encoding a SARS-CoV-2 S protein from a Wuhan strain), (ii) a 60 ug doseof BNT162b2, (iii) a 30 ug dose of RNA encoding a SARS-CoV-2 S proteinhaving mutations characteristic of an Omicron BA.1 variant (“BNT162b2OMI”), (iii) a 60 ug dose of BNT162b2 OMI, (iv) a 30 ug dose of abivalent vaccine, comprising 15 ug of BNT162b2 and 15 ug of BNT162b2 OMI(“Bivalent”), or (v) a 60 ug dose of a bivalent vaccine, comprising 30ug of BNT162b2 and 30 ug of BNT162b2 OMI. Geometric mean titer (GMT) ofneutralization antibodies were measured in serum from subjects collected1 month after administration of a 4th dose (GMT indicated above eachbar). Shown below the x-axis is the geometric fold rise (GMFR) in titerfrom before administration of a 4^(th) dose (pre-vax) to 1 month afteradministration of a 4^(th) dose (1MPD) for each patient group.Neutralization data was obtained using a neutralization assay, with aviral particle containing a SARS-CoV-2 S protein having mutationscharacteristic of a Omicron BA.1 variant.

FIG. 26 . Omicron BA.1 breakthrough infection of BNT162b2 double- andtriple-vaccinated individuals induces broad neutralization of OmicronBA.1, BA.2 and other VOCs, but to a lesser extent against BA.4 and BA.5.This figure is an extension of FIG. 16 , including data neutralizingactivity against Omicron BA.4 and BA.5. As described in FIG. 16 , serumwas tested in duplicate; 50% pseudovirus neutralization (pVN₅₀)geometric mean titers (GMTs) (in A and B), and the geometric mean ratioof SARS-CoV-2 variants of concern (VOCs) and SARS-CoV-1 pVN₅₀ GMTsnormalized against Wuhan pVN₅₀ GMTs (in C) were plotted. For titervalues below the limit of detection (LOD), LOD/2 values were plotted.Values above violin plots represent the group GMTs. The nonparametricFriedman test with Dunn's multiple comparisons correction was used tocompare Wuhan neutralizing group GMTs with titers against the indicatedvariants and SARS-CoV-1. Multiplicity adjusted p values are shown. (A)pVN₅₀ GMTs against Wuhan, VOC and SARS-CoV-1 pseudovirus in patients whoreceived two doses or three doses of BNT162b2. (B) pVN₅₀ GMTs againstWuhan, VOC and SARS-CoV-1 pseudovirus in patients who received two dosesor three doses of BNT162b2 and who have been previously infected with anOmicron BA.1 variant of SARS-CoV-2. (C) Group geometric mean ratios with95% confidence intervals for all cohorts shown in (A) and (B).

FIG. 27 . Omicron BA.1 breakthrough infection of individuals vaccinatedwith other approved COVID-19 vaccines or mixed regimens results inimmune sera that broadly neutralize Omicron BA.1, BA.2 and other VOCs,but to a lesser extent against BA.4 and BA.5. This figure is anextension of FIG. 19 , including data neutralizing activity againstOmicron BA.4 and BA.5. As described in FIG. 19 , serum was tested induplicate; individual 50% pseudovirus neutralization (pVN₅₀) geometricmean titers (GMTs) against SARS-CoV-2 Wuhan, Alpha, Beta, Delta andOmicron BA.1, BA.2 and BA.4/5 variants, plus SARS-CoV-1 were plotted.For titer values below the limit of detection (LOD), LOD/2 values wereplotted. Values above violin plots represent group GMTs. Thenonparametric Friedman test with Dunn's multiple comparisons correctionwas used to compare Wuhan neutralizing group GMTs with titers againstthe indicated variants and SARS-CoV-1. Multiplicity-adjusted p valuesare shown.

FIG. 28 . Sequences of RBDs of SARS-COV-2 Wuhan strain and variantsthereof (SEQ ID NO: 129-133). Variant-specific amino acid alterationsare indicated in bold red font, with the original Wuhan amino acidhighlighted in bold blue font.

FIG. 29 . Cohorts and sampling for the study described in Example 14. Aschematic is shown for testing immune responses in triple-vaccinatedpatients who are (i) Omicron naïve, (ii) have been infected with anOmicron BA.1 variant, or (iii) have been infected with an Omicron BA.2variant. Blood samples were drawn from three cohorts: Omicron-naïveindividuals triple-vaccinated with BNT162b2 (BNT162b2³, green), andindividuals vaccinated with homologous or heterologous three dosesregimens that subsequently had either a breakthrough infection withOmicron at a time of BA.1 dominance (November 2021 to January 2022; allVax+BA.1, purple) or at a time of BA.2 dominance (March to May 2022; allVax+BA.2, blue) in Germany. Sera (droplet) were isolated in theOmicron-naïve cohort at the time-point indicated following their mostrecent vaccination; for convalescent cohorts, the time from their mostrecent vaccination to Omicron infection, and infection to serumisolation are indicated. All values specified as median-range. Serumneutralizing capacity was assessed using a pseudovirus neutralizationtest.

FIG. 30 . 50% pseudovirus neutralization (pVN₅₀) geometric mean titers(GMTs) from the BNT162b2³ and All Vax+Omi BA.1 breakthrough infectioncohorts. Serum was drawn from Omicron-naïve BNT162b2 triple-vaccinatedindividuals (BNT162b2³, circles) at 28 days after the third dose, andfrom vaccinated individuals with subsequent Omicron BA.1 breakthroughinfection (all Vax+Omi BA.1, triangles) at a median 43 dayspost-infection. 50% pseudovirus neutralization (pVN₅₀) geometric meantiters (GMTs) for Omicron-naïve individuals are plotted in (A) and forBA.1 breakthrough infected individuals in (B). This data was previouslypublished in Quandt et al. (“Omicron BA.1 breakthrough infection drivescross-variant neutralization and memory B cell formation againstconserved epitopes.” Science immunology, eabq2427 (2022),doi:10.1126/sciimmunol.abq2427), except for BA.2.12.1 neutralizationdata. Serum was tested in duplicate. For titer values below the limit ofdetection (LOD), LOD/2 values were plotted. Values above violin plotsrepresent group GMTs. The non-parametric Friedman test with Dunn'smultiple comparisons correction was used to compare Wuhan neutralizinggroup GMTs with titers against the indicated variants and SARS-CoV-1.Multiplicity-adjusted p values are shown.

FIG. 31 . Omicron BA.2 breakthrough infection of previously vaccinatedindividuals refocuses neutralization against Omicron BA.2 and theBA.2-derived subvariants BA.2.12.1 and BA.4/BA.5. Serum was drawn fromBNT162b2 triple-vaccinated individuals with subsequent Omicron BA.1breakthrough infection at a median 44 days post-infection (BNT162b2³+OmiBA.1, triangles), and from BNT162b2 triple-vaccinated individuals withsubsequent Omicron BA.2 breakthrough infection at 38 days post-infection(BNT162b2³+Omi BA.2, squares). 50% pseudovirus neutralization (pVN₅₀)geometric mean titers (GMTs) (in A, B), and the geometric mean ratio ofSARS-CoV-2 variants of concern (VOCs) and SARS-CoV-1 pVN₅₀ GMTsnormalized against Wuhan pVN50 GMTs (in C) were plotted. pVN₅₀ GMT andgeometric mean ratio data for Omicron-naïve BNT162b2 triple-vaccinatedindividuals (BNT162b2³, circles) and BNT162b2 triple-vaccinatedindividuals with Omicron BA.1 breakthrough infection was previouslypublished in Quandt et al. (“Omicron BA.1 breakthrough infection drivescross-variant neutralization and memory B cell formation againstconserved epitopes.” Science immunology, eabq2427 (2022),doi:10.1126/sciimmunol.abq2427), except for BA.2.12.1 neutralizationdata. Serum was tested in duplicate. For titer values below the limit ofdetection (LOD), LOD/2 values were plotted. Values above violin plotsrepresent group GMTs. The non-parametric Friedman test with Dunn'smultiple comparisons correction was used to compare Wuhan neutralizinggroup GMTs with titers against the indicated variants and SARS-CoV-1.Multiplicity-adjusted p values are shown. (A, B) pVN₅₀ GMTs againstWuhan, VOC and SARS-CoV-1 pseudovirus. (C) Group geometric mean ratioswith 95% confidence intervals.

FIG. 32 . Characteristics of SARS-CoV-2 S glycoproteins used in theVSV-SARS-CoV-2 pseudovirus based neutralization assays. The sequence ofthe Wuhan-Hu-1 isolate SARS-CoV-2 S glycoprotein (GenBank: QHD43416.1)was used as reference. Amino acid positions, amino acid descriptions(one letter code) and kind of mutations (substitutions, deletions,insertions) are indicated. NTD, N-terminal domain; RBD, Receptor-bindingdomain, Δ, deletion; ins, insertion; *, Cytoplasmic domain truncated forthe C-terminal 19 amino acids.

FIG. 33 . Alterations on the spike glycoprotein amino acid sequence ofSARS-CoV-2 Omicron sub-lineages. Amino acid positions, amino aciddescriptions (one letter code) and kind of mutations substitutions,deletions, insertions) are indicated. White letters in boxes indicatethe amino acid substitution per sub-lineage; Δ, deletion; ins,insertion; NTD, N-terminal domain; RBD, receptor-binding domain.

FIG. 34 . Immunization protocol for studies with VOC boosters. BALB/cmice were immunized according to the indicated schedule with two doses(1 ug each) of the original BNT162b2 vaccine, followed by at least onedose (1 ug total) of a monovalent, bivalent, or trivalent booster doseof either: (a) the original BNT162b2 (“BNT162b2”); (b) BNT162b2 OMI BA.1(“OMI BA.1”); (c) BNT162b2 OMI BA.4/5 (“OMI BA.4/5”); or a combinationthereof.

FIG. 35 . Baseline grouped neutralizing GMTs. Sera drawn from miceimmunized as depicted in FIG. 34 (day 104, pre-boost) were assessed forgeometric mean titers of neutralizing antibodies against variousstrains. Data are presented grouped by cohort.

FIG. 36 . Baseline staggered neutralizing GMTs. Sera drawn from miceimmunized as depicted in FIG. 34 (day 104, pre-boost) were assessed forgeometric mean titers of neutralizing antibodies against variousstrains. Data are presented in staggered format (i.e., by strain againstwhich neutralization was assessed).

FIG. 37 . Baseline cross-neutralization. Sera drawn from mice immunizedas depicted in FIG. 34 (day 104, pre-boost) were assessed for geometricmean titers of neutralizing antibodies against various strains.Cross-neutralization results are presented as calculated variant/Wuhanreference GMT Ratios.

FIG. 38 . Post-boost geometric mean fold increase in GMTs. Sera drawnfrom mice immunized as depicted in FIG. 34 (day 111, 7-days post-boost)were assessed for geometric mean fold increase in GMT of neutralizingantibodies against various strains.

FIG. 39 . Post-boost grouped neutralizing GMTs. Sera drawn from miceimmunized as depicted in FIG. 34 (day 111, 7-days post-boost) wereassessed for geometric mean fold increase in GMT of neutralizingantibodies against various strains. Data are presented grouped bycohort.

FIG. 40 . Post-boost cross-neutralization. Sera drawn from miceimmunized as depicted in FIG. 34 (day 111, 7-days post-boost) wereassessed for geometric mean fold increase in GMT of neutralizingantibodies against various strains. Cross-neutralization results arepresented as calculated variant/Wuhan reference GMT Ratios.

FIG. 41 . Cohorts and sampling. Serum samples (droplet) were drawn fromthree cohorts: individuals triple-vaccinated with BNT162b2 that wereSARS-CoV-2-naïve at the time of sampling (BNT162b2³), and fromindividuals vaccinated with three doses of mRNA COVID-19 vaccine(BNT162b2/mRNA-1273 homologous or heterologous regimens) whosubsequently had a breakthrough infection with Omicron either at a timeof BA.1 dominance (November 2021 to January 2022; mRNA-Vax³+BA.1) or ata time of BA.2 dominance (March to May 2022; mRNA-Vax³+BA.2). Forconvalescent cohorts, relevant intervals between key events such as themost recent vaccination, SARS-CoV-2 infection, and serum isolation areindicated. All values specified as median-range. N/A, not applicable.

FIG. 42 . Omicron BA.2 breakthrough infection of triple mRNA vaccinatedindividuals induces broad neutralization of SARS-CoV-2 variantpseudoviruses including Omicron BA.4/5. Cohorts and serum sampling asdescribed in FIG. 41 . (A) 50% pseudovirus neutralization (pVN₅₀)geometric mean titers (GMTs) against the indicated SARS-CoV-2 variantsof concern (VOCs) or SARS-CoV-1 pseudoviruses. Values above violin plotsrepresent group geometric mean titers (GMTs). BNT162b2³ indicates triplevaccinated, SARS-CoV-2-naïve individuals; mRNA-Vax³+BA.1 indicatestriple-vaccinated individuals who subsequently had a breakthroughinfection with a Omicron BA.1 variant; and mRNA-Vax³+BA.2 indicatestriple vaccinated individuals who subsequently had a breakthroughinfection with an Omicron BA.2 variant. (B) SARS-CoV-2 variant ofconcern (VOC) pVN₅₀ GMTs normalized against the wild-type strain pVN₅₀GMT (ratio VOC to wild-type). Group geometric mean ratios with 95%confidence intervals are shown in the graph and listed in the table.Serum was tested in duplicate. For titer values below the limit ofdetection (LOD), LOD/2 values were plotted. The non-parametric Friedmantest with Dunn's multiple comparisons correction was used to compare thewild-type strain neutralizing group GMTs with titers against theindicated variants and SARS-CoV-1. Multiplicity-adjusted p values areshown.

FIG. 43 . Omicron BA.2 breakthrough infection of previously vaccinatedindividuals induces broad neutralization of authentic live SARS-CoV-2variants including Omicron BA.4/5. Cohorts and serum sampling asdescribed in FIG. 41 . (A) 50% virus neutralization (VN₅₀) geometricmean titers (GMTs) against the indicated SARS-CoV-2 variants of concern(VOCs). Values listed above violin plots represent group GMTs. (B)SARS-CoV-2 VOC VN₅₀ GMTs normalized against the wild-type strain VN₅₀GMT (ratio VOC to wild-type). Group geometric mean ratios with 95%confidence intervals are shown in the graph and listed in the table.Serum was tested in duplicate. For titer values below the limit ofdetection (LOD), LOD/2 values were plotted. The non-parametric Friedmantest with Dunn's multiple comparisons correction was used to compare thewild-type strain neutralizing group GMTs with titers against theindicated variants and SARS-CoV-1. Multiplicity-adjusted p values areshown.

FIG. 44 . Neutralization of Omicron BA.2 and BA.4/5 by sera of triplemRNA vaccinated BA.2 convalescent individuals is mediated to a largeextent by NTD-targeting antibodies. Cohorts and serum sampling asdescribed in FIG. 41 . (A) Serum samples (n=6 per cohort) were depletedof RBD- or NTD-binding antibodies. Relative neutralizing activity ofRBD- and NTD-depleted sera (pVN₅₀ titers of undepleted control sera wereset to 100%) against the wild-type strain, BA.1, BA.2, and BA.4/5 wascalculated and group geometric mean with 95% confidence intervals areshown. (B) 50% pseudovirus neutralization (pVN₅₀) geometric mean titers(GMTs) against Omicron BA.4/5 and Omicron BA.1-BA.4/5 hybridpseudoviruses. Numbers above plots indicate group geometric mean titers(GMTs) and fold-change in GMTs between BA.4/5 and the hybridpseudovirus. For titer values below the limit of detection (LOD), LOD/2values are plotted.

FIG. 45 . Omicron BA.2 breakthrough infection of BNT162b2triple-vaccinated individuals induces broad neutralization of VOCsincluding Omicron BA.4/BA.5. Cohorts and serum sampling as described inFIG. 41 . (A)-(B) 50% pseudovirus neutralization (pVN₅₀) geometric meantiters (GMTs) against the indicated SARS-CoV-2 variants of concern(VOCs) or SARS-CoV-1 pseudoviruses. Values listed above violin plotsrepresent group GMTs. (C) The ratio of SARS-CoV-2 VOC pVN₅₀ GMTsnormalized against the wild-type strain pVN₅₀ GMT. Geometric mean ratiosfor the Omicron BA.2 breakthrough infected cohort were compared toBNT162b2³ and BNT162b2³+BA.1. Group geometric mean ratios with 95%confidence intervals are shown. (D)-(E) 50% virus neutralization (VN₅₀)GMTs for BNT162b2³+BA.2 and BNT162b2³+BA.1. Values listed above violinplots represent group GMTs. (F) The ratio of SARS-CoV-2 VOC GMTsnormalized against the wild-type strain VN₅₀ GMT. Serum was tested induplicate. For titer values below the limit of detection (LOD), LOD/2values are plotted. The non-parametric Friedman test with Dunn'smultiple comparisons correction was used to compare the group GMTagainst the wild-type strain with group GMTs against the indicatedvariants and SARS-CoV-1. Multiplicity-adjusted p values are shown.

FIG. 46 . 50% pseudovirus neutralization (pVN₅₀) correlates with 50%live SARS-CoV-2 neutralization (VN₅₀) titer data. Nonparametric Spearmancorrelation of VSV-SARS-CoV-2 pVN₅₀ with live SARS-CoV-2 VN₅₀ titers forn=45 serum samples drawn from SARS-CoV-2-naïve BNT162b2triple-vaccinated individuals (BNT162b2³; n=18) after the third dose,from triple mRNA vaccinated individuals with subsequent Omicron BA.1breakthrough infection (mRNA-Vax³+BA.1; n=14) post-infection, and fromtriple mRNA vaccinated individuals with subsequent Omicron BA.2breakthrough infection (mRNA-Vax³+BA.2; n=13) post-infection.Correlations are plotted per SARS-CoV-2 variant. Correlation coefficientr, two-tailed P values and the linear equation are given.

FIG. 47 . RBD-binding and NTD-binding antibodies can be depleted fromhuman serum. Serum was drawn from SARS-CoV-2-naïve BNT162b2triple-vaccinated individuals (BNT162b2³; n=6), and from triple RNAvaccinated individuals with Omicron BA.1 (mRNA-Vax³+BA.1; n=6) orOmicron BA.2 breakthrough infection (mRNA-Vax³+BA.2; n=6). Magnetic beadtechnology was used for depleting serum of RBD- or NTD-bindingantibodies, or for mock depleting. (A) Schematic of antibody depletionfrom serum. (B) The relative concentration of RBD-binding andNTD-binding antibodies was determined by a multiplexedelectrochemiluminescence immunoassay. The relative decrease in antibodyconcentrations in depleted compared to mock-depleted sera are shown.Numbers above graph depict geometric mean reduction within groups.

FIG. 48 . Characterization of SARS-CoV-2 S glycoproteins used in theassays based on live authentic SARS-CoV-2. The sequence of theWuhan-Hu-1 isolate SARS-CoV-2 S glycoprotein (GenBank: QHD43416.1) wasused as reference. Amino acid positions, amino acid descriptions (oneletter code) and kind of alterations (substitutions, deletions,insertions) are indicated. NTD, N-terminal domain; RBD, Receptor-bindingdomain, Δ, deletion; ins, insertion; *, Cytoplasmic domain truncated forthe C-terminal 19 amino acids.

FIG. 49 . BA.4/5-Breakthrough Infection and BA.4/5-Booster Study Design.(a) The effect of Omicron BA.4/BA.5 breakthrough infection on serumneutralizing activity was evaluated in individuals vaccinated with threedoses of mRNA COVID-19 vaccine (BNT162b2/mRNA-1273 homologous orheterologous regimens) who subsequently experienced an infection withOmicron BA.4 or BA.5. The intervals between vaccination, breakthroughinfection and sampling are indicated as median/range. (b) Effects ofOmicron BA.4/BA.5-adapted booster vaccines on serum neutralizingactivity was investigated in mice vaccinated twice (with the twovaccines administered 21-days apart) with BNT162b2, followed by abooster dose of BA.4/BA.5-adapted vaccines 3.5 months later.Neutralizing activity was assessed before (pre-D3) and 7, 21, and 35days after the booster dose (d7D3, d21D3, d35D3, respectively). (c) Theeffects of Omicron BA.4/BA.5-adapted vaccines on serum neutralizingactivity were investigated in vaccine-naïve mice vaccinated twice (withthe two vaccines administered 21-days apart) with BA.4/BA.5-adaptedvaccines. Neutralizing activity was assessed 14 days afteradministration of the second dose (d14D2).

FIG. 50 . Omicron BA.4/BA.5 breakthrough infection of triple mRNAvaccinated Individuals mediates pan-Omicron neutralization. Cohorts andserum sampling as described in FIG. 53 . (a) 50% pseudovirusneutralization (pVN₅₀) geometric mean titers (GMTs) in sera ofmRNA-Vax³+BA.4/BA.5 against the indicated SARS-CoV-2 variants of concern(VOCs) or SARS-CoV-1 pseudoviruses. Values above bars represent groupGMTs. (b) SARS-CoV-2 VOC pVN₅₀ GMTs normalized against the wild-typestrain pVN₅₀ GMT (ratio VOC to wild-type) of mRNA-Vax³+BA.4/BA.5 and thereference cohorts as outlined in FIG. 53 . Group geometric mean ratioswith 95% confidence intervals are shown. (c) 50% virus neutralization(VN₅₀) GMTs in sera of mRNA-Vax³+BA.4/BA.5 against the indicatedSARS-CoV-2 VOCs. (d) SARS-CoV-2 VOC VN₅₀ GMTs normalized against thewild-type strain VN₅₀ GMT (ratio VOC to wild-type). Serum was tested induplicate. For titer values below the limit of detection (LOD), LOD/2values were plotted. The non-parametric Friedman test with Dunn'smultiple comparisons correction was used to compare the wild-type strainneutralizing group GMTs with titers against the indicated variants andSARS-CoV-1. Multiplicity-adjusted p values are shown.

FIG. 51 . Booster immunization with an Omicron BA.4/BA.5 S glycoproteinadapted RNA-vaccine mediates pan-Omicron neutralization indouble-vaccinated mice. BALB/c mice (n=8) were injected intramuscularlywith two doses of 1 μg BNT162b2 21 days apart, and a third dose ofeither BNT162b2 (1 μg) or the indicated monovalent (1 μg) or bivalent(0.5 μg each) Omicron BA.1 or BA.4/5-adapted vaccines 104 days after thefirst vaccination. (a) 50% pseudovirus neutralization (pVN₅₀) geometricmean titers (GMTs) against the indicated SARS-CoV-2 variants of concern(VOCs) in sera collected 21 days after the third vaccination (d21D3).Values above bars represent group GMTs. (b) Geometric mean fold-increase(GMFI) of pVN₅₀ titers on d21D3 relative to baseline titers before thethird vaccination. Values above bars represent group GMFIs. (c)SARS-CoV-2 VOC pVN₅₀ GMTs normalized against the wild-type strain pVN₅₀GMT (ratio VOC to wild-type). Group geometric mean ratios are shown. (d)GMFI of pVN₅₀ titers against BA.1 and BA.4/5 over time relative tobaseline titers before the booster vaccination with BNT162b2/BA.1 orBNT162b2/BA.4/5 bivalent vaccines. For titer values below the limit ofdetection (LOD), LOD/2 values were plotted. Error bars represent 95%confidence intervals (e) 50% virus neutralization (VN₅₀) GMTs againstthe indicated SARS-CoV-2 VOCs on d21D3. Values above bars representgroup GMTs. Error bars represent 95% confidence interval. (f) SARS-CoV-2VOC VN₅₀ GMTs normalized against the wild-type strain VN₅₀ GMT (ratioVOC to wild-type). Group geometric mean ratios are shown. Serum wastested in duplicate. For titer values below the limit of detection(LOD), LOD/2 values were plotted. Error bars represent 95% confidenceintervals.

FIG. 52 . Immunization with an Omicron BA.4/BA.5 S glycoproteinsupplemented BNT162b2 mRNA vaccine drives pan-Omicron neutralization inpreviously unvaccinated mice.

Vaccine-naïve BALB/c mice (n=5) were injected intramuscularly with twodoses of either BNT162b2 (1 μg) or the indicated monovalent (1 μg) orbivalent (0.5 μg each) Omicron BA.1 or BA.4/5-adapted vaccines, 21 daysapart. (a) 50% pseudovirus neutralization (pVN₅₀) geometric mean titers(GMTs) against the indicated SARS-CoV-2 variants of concern (VOCs) insera collected 14 days after the second vaccination (d14D2). Valuesabove bars represent group GMTs. Error bars represent 95% confidenceinterval. Serum was tested in duplicate. For titer values below thelimit of detection (LOD), LOD/2 values were plotted. “Wild-type” refersto neutralization titers against a pseudovirus comprising a SARS-CoV-2 Sprotein of the original Wuhan variant.

FIG. 53 . Cohorts and sampling. Serum samples (droplet) were drawn fromfour cohorts: individuals vaccinated with three doses of mRNA COVID-19vaccine (BNT162b2/mRNA-1273 homologous or heterologous regimens) whosubsequently had a breakthrough infection with Omicron BA.4/BA.5(mRNA-Vax³+BA.4/BA.5). The breakthrough infections occurred at a time ofBA.4/BA.5 dominance and/or were variant-confirmed by genome sequencing.Three cohorts were included in the study as references: triple-mRNAvaccinated individuals who experienced breakthrough infection at a timeof either BA.2 dominance (March to May 2022; mRNA-Vax³+BA.2), or BA.1dominance (November 2021 to January 2022; mRNA-Vax³+BA.1), orindividuals triple-vaccinated with BNT162b2 that were SARS-CoV-2-naïveat the time of sampling (BNT162b2³). For convalescent cohorts, relevantintervals between key events such as the most recent vaccination,SARS-CoV-2 infection, and serum isolation are indicated. All valuesspecified as median-range. N/A, not applicable.

FIG. 54 . Design of Omicron-adapted vaccines. Characterization ofSARS-CoV-2 S glycoproteins encoded by variant-specific RNA vaccines(e.g., in some embodiments mRNA vaccines). The sequence of theWuhan-Hu-1 isolate SARS-CoV-2 S glycoprotein (GenBank: QHD43416.1) wasused as reference. Amino acid positions, amino acid descriptions (oneletter code) and type of alterations (substitutions, deletions,insertions) are indicated. NTD, N-terminal domain; RBD, Receptor-bindingdomain, Δ, deletion; ins, insertion. In some embodiments, variantspecific vaccines further comprise mutations that stabilize a pre-fusionconformation (e.g., proline mutations at positions corresponding toresidues 986 and 987 of SEQ ID NO: 1) and/or do not comprise aC-terminal truncation.

FIG. 55 . Omicron-specific vaccines exhibit comparable RNA purity andintegrity, and In vitro expression of antigens. (a) Liquid capillaryelectropherograms of in vitro transcribed samples. (b) Surfaceexpression of BNT162b2 and Omicron-adapted vaccines in HEK293T cellsmeasured using mFc-tagged human ACE-2 as a detection reagent withsubsequent analysis in flow cytometry. HEK293T cells were transfectedwith BNT162b2 or Omicron-adapted vaccines formulated as lipidnanoparticles or vaccine RNAs mixed with a commercial transfectionreagent, or no vaccine/RNA (non-transfected). Heights of bars indicatethe means of technical replicates.

FIG. 56 . Breadth and magnitude of neutralizing activity againstSARS-CoV-2 variants are comparable in BNT162b2-vaccinated mice prior tobooster vaccination. BALB/c mice were injected intramuscularly with twodoses of 1 μg BNT162b2, administered 21 days apart. Mice were allocatedto groups (n=8) prior to administration with a booster dose of theindicated vaccines. Serum was collected from mice on day 104 after thefirst vaccination, before booster vaccines were injected. (a) 50%pseudovirus neutralization (pVN₅₀) geometric mean titers (GMTs) againstthe indicated SARS-CoV-2 variants of concern (VOCs). Values above barsrepresent group GMTs. Error bars represent 95% confidence intervals. (b)SARS-CoV-2 VOC pVN₅₀ GMTs normalized against the wild-type strain pVN₅₀GMT (ratio VOC to wild-type). Group geometric mean ratios are shown.Serum was tested in duplicate. For titer values below the limit ofdetection (LOD), LOD/2 values were plotted.

FIG. 57 . Booster immunization with an Omicron BA.4/BA.5 S glycoproteinadapted mRNA vaccine mediates pan-Omicron neutralization in mice 7 daysafter administering the booster. BALB/c mice (n=8) were injectedintramuscularly with two doses of 1 μg BNT162b2 (with a 21-day intervalbetween the two doses), and a third dose of either BNT162b2 (1 μg) orthe indicated monovalent (1 μg) or bivalent (0.5 Vg each) Omicron BA.1or BA.4/5-adapted vaccines 104 days after the first vaccination. (a) 50%pseudovirus neutralization (pVN₅₀) geometric mean titers (GMTs) againstthe indicated SARS-CoV-2 variants of concern (VOCs) in sera collected 7days after the third vaccination (d7D3). Values above bars representgroup GMTs. (b) Geometric mean fold-increase (GMFI) of pVN₅₀ titers ond7D3 relative to baseline titers before the third vaccination. Valuesabove bars represent group GMFIs. (c) SARS-CoV-2 VOC pVN₅₀ GMTsnormalized against the wild-type strain pVN₅₀ GMT (ratio VOC towild-type). Group geometric mean ratios are shown. Serum was tested induplicate. For titer values below the limit of detection (LOD), LOD/2values were plotted. Error bars represent 95% confidence intervals.

FIG. 58 . Booster immunization with an Omicron BA.4/BA.5 S glycoproteinadapted vaccine mediates pan-Omicron neutralization in mice 35 daysafter the booster. BALB/c mice (n=8) were injected intramuscularly withtwo doses of 1 μg BNT162b2 (with a 21-day interval between the twodoses), and a third dose of either BNT162b2 (1 μg) or the indicatedmonovalent (1 μg) or bivalent (0.5 μg each) Omicron BA.1 orBA.4/5-adapted vaccines 104 days after the first vaccination. (a) 50%pseudovirus neutralization (pVN₅₀) geometric mean titers (GMTs) againstthe indicated SARS-CoV-2 variants of concern (VOCs) in sera collected 35days after the third vaccination (d35D3). Values above bars representgroup GMTs. (b) Geometric mean fold-increase (GMFI) of pVN₅₀ titers ond35D3 relative to baseline titers before the third vaccination. Valuesabove bars represent group GMFIs. (c) SARS-CoV-2 VOC pVN₅₀ GMTsnormalized against the wild-type strain pVN₅₀ GMT (ratio VOC towild-type). Group geometric mean ratios are shown. (d) 50% virusneutralization (VN₅₀) GMTs against the indicated SARS-CoV-2 VOCs ond35D3. Values above bars represent group GMTs. Error bars represent 95%confidence interval. (e) SARS-CoV-2 VOC VN₅₀ GMTs normalized against thewild-type strain VN₅₀ GMT (ratio VOC to wild-type). Group geometric meanratios are shown. Serum was tested in duplicate. For titer values belowthe limit of detection (LOD), LOD/2 values were plotted. Error barsrepresent 95% confidence intervals.

FIG. 59 . Omicron BA.4/BA.5 breakthrough infection of triple mRNAvaccinated individuals induces cross-neutralization of Omicron BA.4.6and BA.2.75. Cohorts and serum sampling as described in FIG. 61 . (a)50% pseudovirus neutralization (pVN₅₀) geometric mean titers (GMTs)against the indicated SARS-CoV-2 wild-type strain or Omicron variants ofconcern (VOCs). Values above bar graphs represent group GMTs. For titervalues below the limit of detection (LOD), LOD/2 values were plotted.The non-parametric Friedman test with Dunn's multiple comparisonscorrection was used to compare neutralizing titers against the OmicronBA.4/BA.5 pseudovirus (which represents currently dominating BA.5) withtiters against the other pseudoviruses. Multiplicity-adjusted p valuesare shown. (b) SARS-CoV-2 VOC pVN₅₀ GMTs normalized against thewild-type strain pVN₅₀ GMT (ratio VOC to wild-type). Group geometricmean ratios with 95% confidence intervals are shown. The non-parametricKruskal-Wallis test with Dunn's multiple comparisons correction was usedto compare the VOC GMT ratios between cohorts. ****, P<0.0001; **,P<0.01; *, P<0.05. Serum was tested in duplicate.

FIG. 60 . Alterations of the spike glycoprotein amino acid sequence ofSARS-CoV-2 Omicron sub-lineages. White letters in boxes indicate theamino acid substitution per sub-lineage; A, deletion; ins, insertion;NTD, N-terminal domain; RBD, receptor-binding domain.

FIG. 61 . Cohorts and sampling. Serum samples were drawn from fivecohorts: SARS-CoV-2-naïve individuals triple-vaccinated with BNT162b2(BNT162b2³) or quadruple-vaccinated with BNT162b2 (BNT162b2⁴), andindividuals with three doses of mRNA COVID-19 vaccine(BNT162b2/mRNA-1273 homologous or heterologous regimens) whosubsequently had a breakthrough infection with Omicron BA.1(mRNA-Vax³+BA.1), with BA.2 (mRNA-Vax³+BA.2) or with BA.4/BA.5(mRNA-Vax³+BA.4/5). Breakthrough infections occurred at a time ofrespective VOC dominance (BA.1: November 2021 to January 2021, BA.2:March to May 2022, BA.4/5: mid-June to mid-July 2022) and/or werevariant confirmed by genome sequencing. For convalescent cohorts,relevant intervals between key events such as the most recentvaccination, SARS-CoV-2 infection, and serum isolation are indicated.All values specified as median-range. N/A, not applicable; ^(§), Serumdraw was performed between 28 to 35 Days after vaccination as perprotocol.

FIG. 62 . Characterization of SARS-CoV-2 S glycoproteins used inVSV-SARS-CoV-2 variant pseudovirus neutralization assays. Mutationpositions shown in reference to the sequence of the Wuhan-Hu-1 isolateSARS-CoV-2 S glycoprotein (GenBank: QHD43416.1). Amino acid positions,amino acid descriptions (one letter code) and kind of alterations(substitutions, deletions, insertions) are indicated. NTD, N-terminaldomain; RBD, Receptor-binding domain, Δ, deletion; ins, insertion; *,Cytoplasmic domain truncated for the C-terminal 19 amino acids.

FIG. 63 . Omicron BA.4/5, BA.4.6, BA.2.75.2, BQ.1.1, and XBB.1neutralizing response with a bivalent (BA.4/5-adapted RNA+BNT162b2) orBNT16b2 monovalent booster. Bar heights and numbers immediately abovebars indicate geometric means of neutralization titers (GMTs). Whiskersindicate 95% CI. Bars labeled as “Bivalent” indicate neutralizationtiters from subjects administered as a 4th dose a bivalent vaccine(comprising RNA encoding a SARS-CoV-2 S protein of a Wuhan strain andRNA encoding a SARS-CoV-2 S protein comprising one or more mutationscharacteristic of a BA.4/5 Omicron variant); bars labeled as“Monovalent” indicate neutralization titers from subjects administeredas a 4th dose a monovalent vaccine (comprising RNA encoding a SARS-CoV-2S protein of a Wuhan strain). FFRNT₅₀s against USA-WA1/2020 spike,BA.4/5-spike, BA.4.6-spike, BA.2.75.2-spike, BQ.1.1-spike, XBB.1-spikeare shown for the bivalent or monovalent booster. “Pre” samplescorrespond to serum samples collected on the day of boosteradministration; “1MPD4” samples correspond to serum samples collectedone month post dose 4 (i.e., one month post booster administration).GMFR corresponds to GMT fold rise, and was calculated as a ratio of1MPD4 GMTs to Pre GMTs. Numbers above GMFRs indicate ratios betweenGMFRs of bivalent and GMFRs of monovalent. (A) FFRNT₅₀s of all subjectsregardless of infection status. p values (two-tailed, Wilcoxonmatched-pairs signed-rank test) for GMFR of bivalent or monovalentbooster against USA-WA1/2020, BA.4/5-spike, BA.4.6-spike,BA.2.75.2-spike, BQ.1.1-spike, XBB.1-spike: all <0.0001. p values(two-tailed, Mann-Whitney test) for GMFR ratios of bivalent tomonovalent booster against USA-WA1/2020, BA.4/5-spike, BA.4.6-spike,BA.2.75.2-spike, BQ.1.1-spike, XBB.1-spike: 0.0061, <0.0001, <0.0001,<0.0001, <0.0001, <0.0001. (B) FFRNT₅₀s of all subjects without evidenceof prior SARS-CoV-2 infection. p values (two-tailed, Wilcoxonmatched-pairs signed-rank test) for GMFR of bivalent booster againstUSA-WA1/2020, BA.4/5-spike, BA.4.6-spike, BA.2.75.2-spike, BQ.1.1-spike,XBB.1-spike: all <0.0001. p values (two-tailed, Wilcoxon matched-pairssigned-rank test) for GMFR of monovalent booster against USA-WA1/2020,BA.4/5-spike, BA.4.6-spike, BA.2.75.2-spike, BQ.1.1-spike, XBB.1-spike:0.013, 0.0006, <0.0001, <0.0001, 0.013, 0.016. p values (two-tailed,Mann-Whitney test) for GMFR ratios of bivalent to monovalent boosteragainst USA-WA1/2020, BA.4/5-spike, BA.4.6-spike, BA.2.75.2-spike,BQ.1.1-spike, XBB.1-spike: 0.035, <0.0001, <0.0001, <0.0001, <0.0001,<0.0001. (C) FFRNT₅₀s of all subjects with evidence of prior SARS-CoV-2infection. p values (two-tailed, Wilcoxon matched-pairs signed-ranktest) for GMFR of bivalent booster against USA-WA1/2020, BA.4/5-spike,BA.4.6-spike, BA.2.75.2-spike, BQ.1.1-spike, XBB.1-spike: all <0.0001. pvalues (two-tailed, Wilcoxon matched-pairs signed-rank test) for GMFR ofmonovalent booster against USA-WA1/2020, BA.4/5-spike, BA.4.6-spike,BA.2.75.2-spike, BQ.1.1-spike, XBB.1-spike: 0.0016, 0.0003, 0.0026,0.0011, <0.0001, <0.0001. p values (two-tailed, Mann-Whitney test) forGMFR ratios of bivalent to monovalent booster against USA-WA1/2020,BA.4/5-spike, BA.4.6-spike, BA.2.75.2-spike, BQ.1.1-spike, XBB.1-spike:0.065, 0.025, 0.0032, 0.0086, 0.0064, 0.0006.

FIG. 64 . Distinct cross-neutralization of Omicron sublineages byvaccine-elicited and convalescent immune sera. (A) 50% pseudovirusneutralization (pVN₅₀) geometric mean titers (GMTs) against theindicated SARS-CoV-2 wild-type strain or Omicron variants of concern(VOCs) in individuals (i) triple- or (ii) quadruple-vaccinated withBNT162b2 that were SARS-CoV-2-naïve at the time of sampling, and fromindividuals vaccinated with three doses of mRNA COVID-19 vaccine(BNT162b2/mRNA-1273 homologous or heterologous regimens) whosubsequently had a breakthrough infection with Omicron at a time of(iii) BA.1 dominance, (iv) BA.2 dominance or (v) BA.4/5 dominance.Values above bar graphs represent group GMTs. For titer values below thelimit of detection (LOD), LOD/2 values were plotted. The non-parametricFriedman test with Dunn's multiple comparisons correction was used tocompare neutralizing titers against the Omicron BA.4/BA.5 pseudovirus(which represents currently dominating BA.5) with titers against theother pseudoviruses. Multiplicity-adjusted p values are shown. (B)SARS-CoV-2 VOC pVN₅₀ GMTs normalized against the wild-type strain pVN₅₀GMT (ratio VOC to wild-type). Group geometric mean ratios with 95%confidence intervals are shown. The non-parametric Kruskal-Wallis testwith Dunn's multiple comparisons correction was used to compare the VOCGMT ratios between cohorts. ****, P<0.0001; ***, P<0.001; **, P<0.01; *,P<0.05. Serum was tested in duplicate.

FIG. 65 . Alterations on the spike glycoprotein amino acid sequence ofSARS-CoV-2 Omicron sub-lineages. Amino acid positions, amino aciddescriptions (one letter code) and types of mutations (substitutions,deletions, insertions) are indicated. White letters in boxes indicatethe amino acid substitution per sub-lineage; A, deletion; ins,insertion; NTD, N-terminal domain; RBD, receptor-binding domain

FIG. 66 . T cell epitope and B cell epitope retention in the S proteinof certain SARS-CoV-2 variants of concern. All potential T cell epitopeswere retrieved from the Immune Epitope Database (IEDB) on Nov. 11, 2022,and the percentages of unaltered S protein linear T cell epitopes (ascompared to epitopes present in BNT162b2—100%) present in each variantstrain were calculated. Percentages of unaltered B cell neutralizationepitopes (NTD and RBD) present in each variant strain compared toBNT162b2 were estimated by an automated Early Warning System (describedin Beguir, Karim, et al., “Early computational detection of potentialhigh risk SARS-CoV-2 variants,” bioRxiv (2021)). (A) Radar chart and (B)line plot analysis of T-cell and neutralizing B cell epitopeconservedness in the S protein across SARS-CoV-2 variants of concern.Percent unaltered T cell epitopes and percent unaltered B cellneutralization epitopes are shown. In (B) numbers on top correspondingto each variant represent number of mutations in spike protein comparingto Wuhan strain. Variants are ranked in ascending order in terms ofmutation number.

DETAILED DESCRIPTION

Although the present disclosure is described in detail below, it is tobe understood that this disclosure is not limited to the particularmethodologies, protocols and reagents described herein as these mayvary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present disclosure which will belimited only by the appended claims. Unless defined otherwise, alltechnical and scientific terms used herein have the same meanings ascommonly understood by one of ordinary skill in the art.

Preferably, the terms used herein are defined as described in “Amultilingual glossary of biotechnological terms: (IUPACRecommendations)”, H. G. W. Leuenberger, B. Nagel, and H. Kölbl, Eds.,Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

The practice of the present disclosure will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, cellbiology, immunology, and recombinant DNA techniques which are explainedin the literature in the field (cf., e.g., Molecular Cloning: ALaboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold SpringHarbor Laboratory Press, Cold Spring Harbor 1989).

In the following, the elements of the present disclosure will bedescribed. These elements are listed with specific embodiments, however,it should be understood that they may be combined in any manner and inany number to create additional embodiments. The variously describedexamples and embodiments should not be construed to limit the presentdisclosure to only the explicitly described embodiments. Thisdescription should be understood to disclose and encompass embodimentswhich combine the explicitly described embodiments with any number ofthe disclosed elements. Furthermore, any permutations and combinationsof all described elements should be considered disclosed by thisdescription unless the context indicates otherwise.

Several documents are cited throughout the text of this specification.Each of the documents cited herein (including all patents, patentapplications, scientific publications, manufacturer's specifications,instructions, etc.), whether supra or infra, are hereby incorporated byreference in their entirety. Nothing herein is to be construed as anadmission that the present disclosure was not entitled to antedate suchdisclosure.

Definitions

In the following, definitions will be provided which apply to allaspects of the present disclosure. The following terms have thefollowing meanings unless otherwise indicated. Any undefined terms havetheir art recognized meanings.

The term “about” means approximately or nearly, and in the context of anumerical value or range set forth herein in one embodiment means ±20%,±10%, ±5%, or ±3% of the numerical value or range recited or claimed.

The terms “a” and “an” and “the” and similar reference used in thecontext of describing the present disclosure (especially in the contextof the claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. Recitation of ranges of values herein is merely intended toserve as a shorthand method of referring individually to each separatevalue falling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wasindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”), provided herein isintended merely to better illustrate the disclosure and does not pose alimitation on the scope of the claims. No language in the specificationshould be construed as indicating any non-claimed element essential tothe practice of the disclosure.

Unless expressly specified otherwise, the term “comprising” is used inthe context of the present document to indicate that further members mayoptionally be present in addition to the members of the list introducedby “comprising”. It is, however, contemplated as a specific embodimentof the present disclosure that the term “comprising” encompasses thepossibility of no further members being present, i.e., for the purposeof this embodiment “comprising” is to be understood as having themeaning of “consisting of” or “consisting essentially of”. Terms such as“reduce”, “decrease”, “inhibit” or “impair” as used herein relate to anoverall reduction or the ability to cause an overall reduction,preferably of at least 5%, at least 10%, at least 20%, at least 50%, atleast 75% or even more, in the level. These terms include a complete oressentially complete inhibition, i.e., a reduction to zero oressentially to zero. Terms such as “increase”, “enhance” or “exceed”preferably relate to an increase or enhancement by at least 10%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 80%, atleast 100%, at least 200%, at least 500%, or even more.

According to the present disclosure, the term “peptide” comprises oligo-and polypeptides and refers to substances which comprise about two ormore, about 3 or more, about 4 or more, about 6 or more, about 8 ormore, about 10 or more, about 13 or more, about 16 or more, about 20 ormore, and up to about 50, about 100 or about 150, consecutive aminoacids linked to one another via peptide bonds. The term “protein” or“polypeptide” refers to large peptides, in particular peptides having atleast about 150 amino acids, but the terms “peptide”, “protein” and“polypeptide” are used herein usually as synonyms.

A “therapeutic protein” has a positive or advantageous effect on acondition or disease state of a subject when provided to the subject ina therapeutically effective amount. In one embodiment, a therapeuticprotein has curative or palliative properties and may be administered toameliorate, relieve, alleviate, reverse, delay onset of or lessen theseverity of one or more symptoms of a disease or disorder. A therapeuticprotein may have prophylactic properties and may be used to delay theonset of a disease or to lessen the severity of such disease orpathological condition. The term “therapeutic protein” includes entireproteins or peptides, and can also refer to therapeutically activefragments thereof. It can also include therapeutically active variantsof a protein. Examples of therapeutically active proteins include, butare not limited to, antigens for vaccination and immunostimulants suchas cytokines.

“Fragment”, with reference to an amino acid sequence (peptide orprotein), relates to a part of an amino acid sequence, i.e. a sequencewhich represents the amino acid sequence shortened at the N-terminusand/or C-terminus. A fragment shortened at the C-terminus (N-terminalfragment) is obtainable e.g. by translation of a truncated open readingframe that lacks the 3′-end of the open reading frame. A fragmentshortened at the N-terminus (C-terminal fragment) is obtainable e.g. bytranslation of a truncated open reading frame that lacks the 5′-end ofthe open reading frame, as long as the truncated open reading framecomprises a start codon that serves to initiate translation. A fragmentof an amino acid sequence comprises e.g. at least 50%, at least 60%, atleast 70%, at least 80%, at least 90% of the amino acid residues from anamino acid sequence. A fragment of an amino acid sequence preferablycomprises at least 6, in particular at least 8, at least 12, at least15, at least 20, at least 30, at least 50, or at least 100 consecutiveamino acids from an amino acid sequence.

By “variant” herein is meant an amino acid sequence that differs from aparent amino acid sequence by virtue of at least one amino acidmodification. The parent amino acid sequence may be a naturallyoccurring or wild type (WT) amino acid sequence, or may be a modifiedversion of a wild type amino acid sequence. Preferably, the variantamino acid sequence has at least one amino acid modification compared tothe parent amino acid sequence, e.g., from 1 to about 20 amino acidmodifications, and preferably from 1 to about 10 or from 1 to aboutamino acid modifications compared to the parent.

By “wild type” or “WT” or “native” herein is meant an amino acidsequence that is found in nature, including allelic variations. A wildtype amino acid sequence, peptide or protein has an amino acid sequencethat has not been intentionally modified.

In some embodiments, the present disclosure refers to a SARS-CoV-2variant that is prevalent and/or rapidly spreading in a relevantjurisdiction. In some embodiments, such variants may be identified basedon publicly available data (e.g., data provided in the GISAID Initiativedatabase: https://www.gisaid.org, and/or data provided by the WorldHealth Organization WHO (e.g., as provided athttps://www.who.int/activities/tracking-SARS-CoV-2-variants). In someembodiments, such a variant refers to a variant disclosed herein.

For the purposes of the present disclosure, “variants” of an amino acidsequence (peptide, protein or polypeptide) comprise amino acid insertionvariants, amino acid addition variants, amino acid deletion variantsand/or amino acid substitution variants. The term “variant” includes allmutants, splice variants, posttranslationally modified variants,conformations, isoforms, allelic variants, species variants, and specieshomologs, in particular those which are naturally occurring. The term“variant” includes, in particular, fragments of an amino acid sequence.

Amino acid insertion variants comprise insertions of single or two ormore amino acids in a particular amino acid sequence. In the case ofamino acid sequence variants having an insertion, one or more amino acidresidues are inserted into a particular site in an amino acid sequence,although random insertion with appropriate screening of the resultingproduct is also possible. Amino acid addition variants comprise amino-and/or carboxy-terminal fusions of one or more amino acids, such as 1,2, 3, 5, 10, 20, 30, 50, or more amino acids. Amino acid deletionvariants are characterized by the removal of one or more amino acidsfrom the sequence, such as by removal of 1, 2, 3, 5, 10, 20, 30, 50, ormore amino acids. The deletions may be in any position of the protein.Amino acid deletion variants that comprise the deletion at theN-terminal and/or C-terminal end of the protein are also calledN-terminal and/or C-terminal truncation variants. Amino acidsubstitution variants are characterized by at least one residue in thesequence being removed and another residue being inserted in its place.Preference is given to the modifications being in positions in the aminoacid sequence which are not conserved between homologous proteins orpeptides and/or to replacing amino acids with other ones having similarproperties. Preferably, amino acid changes in peptide and proteinvariants are conservative amino acid changes, i.e., substitutions ofsimilarly charged or uncharged amino acids. A conservative amino acidchange involves substitution of one of a family of amino acids which arerelated in their side chains. Naturally occurring amino acids aregenerally divided into four families: acidic (aspartate, glutamate),basic (lysine, arginine, histidine), non-polar (alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),and uncharged polar (glycine, asparagine, glutamine, cysteine, serine,threonine, tyrosine) amino acids. Phenylalanine, tryptophan, andtyrosine are sometimes classified jointly as aromatic amino acids. Inone embodiment, conservative amino acid substitutions includesubstitutions within the following groups:

glycine, alanine;valine, isoleucine, leucine;aspartic acid, glutamic acid;asparagine, glutamine;serine, threonine;lysine, arginine; andphenylalanine, tyrosine.

Preferably the degree of similarity, preferably identity between a givenamino acid sequence and an amino acid sequence which is a variant ofsaid given amino acid sequence will be at least about 60%, 70%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99%. The degree of similarity or identity isgiven preferably for an amino acid region which is at least about 10%,at least about 20%, at least about 30%, at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90% or about 100% of the entire length of the referenceamino acid sequence. For example, if the reference amino acid sequenceconsists of 200 amino acids, the degree of similarity or identity isgiven preferably for at least about 20, at least about 40, at leastabout 60, at least about 80, at least about 100, at least about 120, atleast about 140, at least about 160, at least about 180, or about 200amino acids, in some embodiments continuous amino acids. In someembodiments, the degree of similarity or identity is given for theentire length of the reference amino acid sequence. The alignment fordetermining sequence similarity, preferably sequence identity can bedone with art known tools, preferably using the best sequence alignment,for example, using Align, using standard settings, preferablyEMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.“Sequence similarity” indicates the percentage of amino acids thateither are identical or that represent conservative amino acidsubstitutions. “Sequence identity” between two amino acid sequencesindicates the percentage of amino acids that are identical between thesequences. “Sequence identity” between two nucleic acid sequencesindicates the percentage of nucleotides that are identical between thesequences.

The terms “% identical”, “% identity” or similar terms are intended torefer, in particular, to the percentage of nucleotides or amino acidswhich are identical in an optimal alignment between the sequences to becompared. Said percentage is purely statistical, and the differencesbetween the two sequences may be but are not necessarily randomlydistributed over the entire length of the sequences to be compared.Comparisons of two sequences are usually carried out by comparing thesequences, after optimal alignment, with respect to a segment or “windowof comparison”, in order to identify local regions of correspondingsequences. The optimal alignment for a comparison may be carried outmanually or with the aid of the local homology algorithm by Smith andWaterman, 1981, Ads App. Math. 2, 482, with the aid of the localhomology algorithm by Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443,with the aid of the similarity search algorithm by Pearson and Lipman,1988, Proc. Natl Acad. Sci. USA 88, 2444, or with the aid of computerprograms using said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST Nand TFASTA in Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Drive, Madison, Wis.). In some embodiments, percentidentity of two sequences is determined using the BLASTN or BLASTPalgorithm, as available on the United States National Center forBiotechnology Information (NCBI) website (e.g., atblast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq).In some embodiments, the algorithm parameters used for BLASTN algorithmon the NCBI website include: (i) Expect Threshold set to 10; (ii) WordSize set to 28; (iii) Max matches in a query range set to 0; (iv)Match/Mismatch Scores set to 1, -2; (v) Gap Costs set to Linear; and(vi) the filter for low complexity regions being used. In someembodiments, the algorithm parameters used for BLASTP algorithm on theNCBI website include: (i) Expect Threshold set to 10; (ii) Word Size setto 3; (iii) Max matches in a query range set to 0; (iv) Matrix set toBLOSUM62; (v) Gap Costs set to Existence: 11 Extension: 1; and (vi)conditional compositional score matrix adjustment.

Percentage identity is obtained by determining the number of identicalpositions at which the sequences to be compared correspond, dividingthis number by the number of positions compared (e.g., the number ofpositions in the reference sequence) and multiplying this result by 100.

In some embodiments, the degree of similarity or identity is given for aregion which is at least about 50%, at least about 60%, at least about70%, at least about 80%, at least about 90% or about 100% of the entirelength of the reference sequence. For example, if the reference nucleicacid sequence consists of 200 nucleotides, the degree of identity isgiven for at least about 100, at least about 120, at least about 140, atleast about 160, at least about 180, or about 200 nucleotides, in someembodiments continuous nucleotides. In some embodiments, the degree ofsimilarity or identity is given for the entire length of the referencesequence. Homologous amino acid sequences exhibit according to thepresent disclosure at least 40%, in particular at least 50%, at least60%, at least 70%, at least 80%, at least 90% and preferably at least95%, at least 98 or at least 99% identity of the amino acid residues.

The amino acid sequence variants described herein may readily beprepared by the skilled person, for example, by recombinant DNAmanipulation. The manipulation of DNA sequences for preparing peptidesor proteins having substitutions, additions, insertions or deletions, isdescribed in detail in Sambrook et al. (1989), for example. Furthermore,the peptides and amino acid variants described herein may be readilyprepared with the aid of known peptide synthesis techniques such as, forexample, by solid phase synthesis and similar methods.

In one embodiment, a fragment or variant of an amino acid sequence(peptide or protein) is preferably a “functional fragment” or“functional variant”. The term “functional fragment” or “functionalvariant” of an amino acid sequence relates to any fragment or variantexhibiting one or more functional properties identical or similar tothose of the amino acid sequence from which it is derived, i.e., it isfunctionally equivalent. With respect to antigens or antigenicsequences, one particular function is one or more immunogenic activitiesdisplayed by the amino acid sequence from which the fragment or variantis derived. The term “functional fragment” or “functional variant”, asused herein, in particular refers to a variant molecule or sequence thatcomprises an amino acid sequence that is altered by one or more aminoacids compared to the amino acid sequence of the parent molecule orsequence and that is still capable of fulfilling one or more of thefunctions of the parent molecule or sequence, e.g., inducing an immuneresponse. In one embodiment, the modifications in the amino acidsequence of the parent molecule or sequence do not significantly affector alter the characteristics of the molecule or sequence. In differentembodiments, the function of the functional fragment or functionalvariant may be reduced but still significantly present, e.g.,immunogenicity of the functional variant may be at least 50%, at least60%, at least 70%, at least 80%, or at least 90% of the parent moleculeor sequence. However, in other embodiments, immunogenicity of thefunctional fragment or functional variant may be enhanced compared tothe parent molecule or sequence.

An amino acid sequence (peptide, protein or polypeptide) “derived from”a designated amino acid sequence (peptide, protein or polypeptide)refers to the origin of the first amino acid sequence. Preferably, theamino acid sequence which is derived from a particular amino acidsequence has an amino acid sequence that is identical, essentiallyidentical or homologous to that particular sequence or a fragmentthereof. Amino acid sequences derived from a particular amino acidsequence may be variants of that particular sequence or a fragmentthereof. For example, it will be understood by one of ordinary skill inthe art that the antigens suitable for use herein may be altered suchthat they vary in sequence from the naturally occurring or nativesequences from which they were derived, while retaining the desirableactivity of the native sequences.

As used herein, an “instructional material” or “instructions” includes apublication, a recording, a diagram, or any other medium of expressionwhich can be used to communicate the usefulness of the compositions andmethods of the present disclosure. The instructional material of the kitof the present disclosure may, for example, be affixed to a containerwhich contains the compositions of the present disclosure or be shippedtogether with a container which contains the compositions.Alternatively, the instructional material may be shipped separately fromthe container with the intention that the instructional material and thecompositions be used cooperatively by the recipient.

“Isolated” means altered or removed from the natural state. For example,a nucleic acid or a peptide naturally present in a living animal is not“isolated”, but the same nucleic acid or peptide partially or completelyseparated from the coexisting materials of its natural state is“isolated”. An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

The term “recombinant” in the context of the present disclosure means“made through genetic engineering”. Preferably, a “recombinant object”such as a recombinant nucleic acid in the context of the presentdisclosure is not occurring naturally.

The term “naturally occurring” as used herein refers to the fact that anobject can be found in nature. For example, a peptide or nucleic acidthat is present in an organism (including viruses) and can be isolatedfrom a source in nature and which has not been intentionally modified byman in the laboratory is naturally occurring.

“Physiological pH” as used herein refers to a pH of about 7.5.

The term “genetic modification” or simply “modification” includes thetransfection of cells with nucleic acid. The term “transfection” relatesto the introduction of nucleic acids, in particular RNA, into a cell.For purposes of the present disclosure, the term “transfection” alsoincludes the introduction of a nucleic acid into a cell or the uptake ofa nucleic acid by such cell, wherein the cell may be present in asubject, e.g., a patient. Thus, according to the present disclosure, acell for transfection of a nucleic acid described herein can be presentin vitro or in vivo, e.g. the cell can form part of an organ, a tissueand/or an organism of a patient. According to the disclosure,transfection can be transient or stable. For some applications oftransfection, it is sufficient if the transfected genetic material isonly transiently expressed. RNA can be transfected into cells totransiently express its coded protein. Since the nucleic acid introducedin the transfection process is usually not integrated into the nucleargenome, the foreign nucleic acid will be diluted through mitosis ordegraded. Cells allowing episomal amplification of nucleic acids greatlyreduce the rate of dilution. If it is desired that the transfectednucleic acid actually remains in the genome of the cell and its daughtercells, a stable transfection must occur. Such stable transfection can beachieved by using virus-based systems or transposon-based systems fortransfection. Generally, nucleic acid encoding antigen is transientlytransfected into cells. RNA can be transfected into cells to transientlyexpress its coded protein.

The term “seroconversion” includes a 24-fold rise from beforevaccination to 1-month post Dose 2.

Coronavirus

Coronaviruses are enveloped, positive-sense, single-stranded RNA ((+)ssRNA) viruses. They have the largest genomes (26-32 kb) among known RNAviruses and are phylogenetically divided into four genera (α, β, γ, andδ), with betacoronaviruses further subdivided into four lineages (A, B,C, and D). Coronaviruses infect a wide range of avian and mammalianspecies, including humans. Some human coronaviruses generally cause mildrespiratory diseases, although severity can be greater in infants, theelderly, and the immunocompromised. Middle East respiratory syndromecoronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus(SARS-CoV), belonging to betacoronavirus lineages C and B, respectively,are highly pathogenic. Both viruses emerged into the human populationfrom animal reservoirs within the last 15 years and caused outbreakswith high case-fatality rates. The outbreak of severe acute respiratorysyndrome coronavirus-2 (SARS-CoV-2) that causes atypical pneumonia(coronavirus disease 2019; COVID-19) has raged in China sincemid-December 2019, and has developed to be a public health emergency ofinternational concern. SARS-CoV-2 (MN908947.3) belongs tobetacoronavirus lineage B. It has at least 70% sequence similarity toSARS-CoV.

In general, coronaviruses have four structural proteins, namely,envelope (E), membrane (M), nucleocapsid (N), and spike (S). The E and Mproteins have important functions in the viral assembly, and the Nprotein is necessary for viral RNA synthesis. The critical glycoproteinS is responsible for virus binding and entry into target cells. The Sprotein is synthesized as a single-chain inactive precursor that iscleaved by furin-like host proteases in the producing cell into twononcovalently associated subunits, S1 and S2. The S1 subunit containsthe receptor-binding domain (RBD), which recognizes the host-cellreceptor. The S2 subunit contains the fusion peptide, two heptadrepeats, and a transmembrane domain, all of which are required tomediate fusion of the viral and host-cell membranes by undergoing alarge conformational rearrangement. The S1 and S2 subunits trimerize toform a large prefusion spike.

The S precursor protein of SARS-CoV-2 can be proteolytically cleavedinto S1 (685 aa) and S2 (588 aa) subunits. The S1 subunit comprises thereceptor-binding domain (RBD), which mediates virus entry into sensitivecells through the host angiotensin-converting enzyme 2 (ACE2) receptor.

Antigen

The present disclosure comprises the use of RNA encoding an amino acidsequence comprising SARS-CoV-2 S protein, an immunogenic variantthereof, or an immunogenic fragment of the SARS-CoV-2 S protein or theimmunogenic variant thereof. Thus, the RNA encodes a peptide or proteincomprising at least an epitope SARS-CoV-2 S protein or an immunogenicvariant thereof for inducing an immune response against coronavirus Sprotein, in particular SARS-CoV-2 S protein in a subject. The amino acidsequence comprising SARS-CoV-2 S protein, an immunogenic variantthereof, or an immunogenic fragment of the SARS-CoV-2 S protein or theimmunogenic variant thereof (i.e., the antigenic peptide or protein) isalso designated herein as “vaccine antigen”, “peptide and proteinantigen”, “antigen molecule” or simply “antigen”. The SARS-CoV-2 Sprotein, an immunogenic variant thereof, or an immunogenic fragment ofthe SARS-CoV-2 S protein or the immunogenic variant thereof is alsodesignated herein as “antigenic peptide or protein” or “antigenicsequence”.

The SARS-CoV-2 coronavirus full length spike (S) protein from the firstdetected SARS-CoV-2 strain (referred to as the Wuhan strain herein)consists of 1273 amino acids and has the amino acid sequence accordingto SEQ ID NO: 1:

(SEQ ID NO: 1) MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTEKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDS EPVLKGVKLHYT

For purposes of the present disclosure, the above sequence is consideredthe wildtype or Wuhan SARS-CoV-2 S protein amino acid sequence. Unlessotherwise indicated, position numberings in a SARS-CoV-2 S protein givenherein are in relation to the amino acid sequence according to SEQ IDNO: 1. One of skill in the art reading the present disclosure willunderstand the corresponding positions in SARS-CoV-2 S protein variantsbased on the position numbering relative to the amino acid sequence ofSEQ ID NO: 1.

In specific embodiments, full length spike (S) protein encoded by an RNAdescribed herein can be modified in such a way that the prototypicalprefusion conformation is stabilized. Certain mutations that stabilize aprefusion confirmation are known in the art, e.g., as disclosed in WO2021243122 A2 and Hsieh, Ching-Lin, et al. (“Structure-based design ofprefusion-stabilized SARS-CoV-2 spikes,” Science 369.6510 (2020):1501-1505), the contents of each which are incorporated by referenceherein in their entirety. In some embodiments, a SARS-CoV-2 S proteinmay be stabilized by introducing one or more proline mutations. In someembodiments, a SARS-CoV-2 S protein comprises a proline substitution atpositions corresponding to residues 986 and/or 987 of SEQ ID NO: 1. Insome embodiments, a SARS-CoV-2 S protein comprises a prolinesubstitution at one or more positions corresponding to residues 817,892, 899, and 942 of SEQ ID NO: 1. In some embodiments, a SARS-CoV-2 Sprotein comprises a proline substitution at positions corresponding toeach of residues 817, 892, 899, and 942 of SEQ ID NO: 1. In someembodiments, a SARS-CoV-2 S protein comprises a proline substitution atpositions corresponding to each of residues 817, 892, 899, 942, 986, and987 of SEQ ID NO: 1.

In some embodiments, stabilization of the prefusion conformation may beobtained by introducing two consecutive proline substitutions atresidues 986 and 987 in the full length spike protein. Specifically,spike (S) protein stabilized protein variants are obtained in a way thatthe amino acid residue at position 986 is exchanged to proline and theamino acid residue at position 987 is also exchanged to proline. In oneembodiment, a SARS-CoV-2 S protein variant wherein the prototypicalprefusion conformation is stabilized comprises the amino acid sequenceshown in SEQ ID NO: 7:

(SEQ ID NO: 7) MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGINGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDS EPVLKGVKLHYT

Those skilled in the art are aware of various spike variants, and/orresources that document them. For example, the following strains, theirSARS-CoV-2 S protein amino acid sequences and, in particular,modifications thereof compared to wildtype SARS-CoV-2 S protein aminoacid sequence, e.g., as compared to SEQ ID NO: 1, are useful herein.

B.1.1.7 (“Variant of Concern 202012/01” (VOC-202012/01)

B.1.1.7 (the “alpha variant”) is a variant of SARS-CoV-2 which was firstdetected in October 2020 during the COVID-19 pandemic in the UnitedKingdom from a sample taken the previous month, and it quickly began tospread by mid-December. It is correlated with a significant increase inthe rate of COVID-19 infection in United Kingdom; this increase isthought to be at least partly because of change N501Y inside the spikeglycoprotein's receptor-binding domain, which is needed for binding toACE2 in human cells. The B.1.1.7 variant is defined by 23 mutations: 13non-synonymous mutations, 4 deletions, and 6 synonymous mutations (i.e.,there are 17 mutations that change proteins and six that do not). Thespike protein changes in B.1.1.7 include deletion 69-70, deletion 144,N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H.

B.1.351 (501.V2)

B.1.351 lineage (the “Beta variant”) and colloquially known as SouthAfrican COVID-19 variant, is a variant of SARS-CoV-2. Preliminaryresults indicate that this variant may have an increasedtransmissibility. The B.1.351 variant is defined by multiple spikeprotein changes including: L18F, D80A, D215G, deletion 242-244, R246I,K417N, E484K, N501Y, D614G and A701V. There are three mutations ofparticular interest in the spike region of the B.1.351 genome: K417N,E484K, N501Y.

B.1.1.298 (Cluster 5)

B.1.1.298 was discovered in North Jutland, Denmark, and is believed tohave been spread from minks to humans via mink farms. Several differentmutations in the spike protein of the virus have been confirmed. Thespecific mutations include deletion 69-70, Y453F, D614G, I692V, M1229I,and optionally S1147L.

P.1 (B.1.1.248)

Lineage B.1.1.248 (the “gamma variant”), known as the Brazil(ian)variant, is one of the variants of SARS-CoV-2 which has been named P.1lineage. P.1 has a number of S-protein modifications [L18F, T20N, P26S,D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I, V1176F] and issimilar in certain key RBD positions (K417, E484, N501) to variantB.1.351 from South Africa.

B.1.427/B.1.429 (CAL.20C)

Lineage B.1.427/B.1.429 (the “epsilon variant”), also known as CAL.20C,is defined by the following modifications in the S-protein: S131, W152C,L452R, and D614G of which the L452R modification is of particularconcern. CDC has listed B.1.427/B.1.429 as “variant of concern”.

B.1.525

B.1.525 (the “eta variant”) carries the same E484K modification as foundin the P.1, and B.1.351 variants, and also carries the same ΔH69/ΔV70deletion as found in B.1.1.7, and B.1.1.298. It also carries themodifications D614G, Q677H and F888L.

B.1.526

B.1.526 (the “iota variant”) was detected as an emerging lineage ofviral isolates in the New York region that shares mutations withpreviously reported variants. The most common sets of spike mutations inthis lineage are L5F, T95I, D253G, E484K, D614G, and A701V.

The following table shows an overview of circulating SARS-CoV-2 strainswhich are VOI/VOC.

TABLE 1 Overview of certain circulating SARS-CoV-2 strains which havebeen/are VOI/VOC Lineage Amino acid substitution P.1 L18F T20N P26SD138Y R190S (BRA) B.1.1.7 ΔH69/V70 ΔY144 (UK) B.1.351 L18F D80A D215G(SA) B.1.1.298 ΔH69/V70 (DK) B.1.427/B.1.429 S13I W152C (CAL) B.1.525ΔH69/V70 B.1.526 L5F T95I (NY) Lineage Amino acid substitution P.1 K417TE484K N501Y D614G H655Y (BRA) B.1.1.7 N501Y A570D D614G (UK) B.1.351Δ242/243 R246I K417N E484K N501Y D614G (SA) B.1.1.298 Y453F D614G (DK)B.1.427/B.1.429 L452R D614G (CAL) B.1.525 E484K D614G B.1.526 D253GE484K D614G (NY) Lineage Amino acid substitution P.1 T1027I V1176F (BRA)B.1.1.7 P681H T716I S982A D1118H (UK) B.1.351 A701V (SA) B.1.1.298 I692VM1229I (DK) B.1.427/B.1.429 (CAL) B.1.525 Q677H F888L B.1.526 A701V (NY)

B.1.1.529

B.1.529 (“Omicron variant”) was first detected in South Africa inNovember 2021. Omicron multiplies around 70 times faster than Deltavariants, and quickly became the dominant strain of SARS-CoV-2worldwide. Since its initial detection, a number of Omicron sublineageshave arisen. Listed below are the current Omicron variants of concern,along with certain characteristic mutations associated with the Sprotein of each. The S protein of BA.4 and BA.5 have the same set ofcharacteristic mutations, which is why the below table has a single rowfor “BA.4 or BA.5”, and why the present disclosure refers to a “BA.4/5”S protein in some embodiments. Similarly, the S proteins of the BA.4.6and BF.7 Omicron variants have the same set of characteristic mutations,which is why the below table has a single row for “BA.4.6 or BF.7”).

TABLE 2 Omicron Variants of Concern and Characteristic mutationsSubvariant Common mutations BA.1 A67V, Δ69-70, T95I, G142D, Δ143-145,Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S,S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G,H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F BA.2 T19I,Δ24-26, A27S, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N,R408S, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H,D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K BA.2.12.1 T19I,Δ24-26, A27S, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N,R408S, K417N, N440K, L452Q, S477N, T478K, E484A, Q493R, Q498R, N501Y,Y505H, D614G, H655Y, N679K, P681H, S704L, N764K, D796Y, Q954H, N969KBA.4 or T19I, Δ24-26, A27S, Δ69/70, G142D, V213G, G339D, S371F, BA.5S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K,E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K,D796Y, Q954H, N969K BA.2.75 T19I, Δ24-26, A27S, G142D, K147E, W152R,F157L, I210V, V213G, G257S, G339H, N354D, S371F, S373P, S375F, T376A,D405N, R408S, K417N, N440K, G446S, N460K, S477N, T478K, E484A, Q498R,N501Y, Y505H D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969KBA.2.75.2 T19I, Δ24-26, A27S, G142D, K147E, W152R, F157L, I210V, V213G,G257S, G339H, R346T, N354D, S371F, S373P, S375F, T376A, D405N, R408S,K417N, N440K, G446S, N460K, S477N, T478K, E484A, F486S, Q498R, N501Y,Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, andD1199N BJ.1 T19I, Δ24-26, A27S, V83A, G142D, Δ144, H146Q, Q183E, V213E,G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N,N440K, V445P, G446S, S477N, T478K, V483A, E484A, F490V, Q493R, Q498R,N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, G798D, Q954H,N969K, and S1003I BA.4.6 or T19I, Δ24-26, A27S, Δ69/70, G142D, V213G,G339D, R346T, BF.7 S371F, S373P, S375F, T376A, D405N, R408S, K417N,N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G,H655Y, N658S, N679K, P681H, N764K, D796Y, Q954H, and N969K XBB T19I,Δ24-26, A27S, V83A, G142D, Δ144, H146Q, Q183E, V213E, G339H, R346T,L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P,G446S, N460K, S477N, T478K, E484A, F486S, F490S, Q493R, Q498R, N501Y,Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K XBB.1T19I, Δ24-26, A27S, V83A, G142D, Δ144, H146Q, Q183E, V213E, G252V,G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N,N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486S, F490S, Q493R,Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H,and N969K XBB.2 T19I, Δ24-26, A27S, V83A, G142D, Δ144, H146Q, Q183E,V213E, D253G, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N,R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486S,F490S, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K,D796Y, Q954H, and N969K XBB.1.3 T19I, Δ24-26, A27S, V83A, G142D, Δ144,H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F,T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K,A484T, F486S, F490S, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K,P681H, N764K, D796Y, Q954H, and N969K BA.2.3.20 T19I, Δ24-26, A27S,G142D, M153T, N164K, V213G, H245N, G257D, G339D, S371F, S373P, S375F,T376A, D405N, R408S, K417N, N440K, K444R, E484R N450D, L452M, N460K,S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K,P681H, N764K, D796Y, Q954H, and N969K BQ.1.1 T19I, Δ24-26, A27S, Δ69/70,G142D, V213G, G339D, R346T, S371F, S373P, S375F, T376A, D405N, R408S,K417N, N440K, K444T, L452R, N460K, S477N, T478K, E484A, F486V, Q498R,N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K

In some embodiments, RNA described herein comprises a nucleotidesequence encoding a SARS-CoV-2 S protein comprising one or moremutations (including, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, or more) that are characteristic of an Omicronvariant (e.g., one or more mutations of an Omicron variant listed inTable 2). In some embodiments, such RNA further comprises one or moremutations that stabilize the S protein in a pre-fusion confirmation(e.g., in some embodiments, such RNA further comprises proline residuesat positions corresponding to residues 986 and 987 of SEQ ID NO: 1). Insome embodiments, an RNA comprises a nucleotide sequence encoding aSARS-CoV-2 S protein comprising one or more mutations (including, e.g.,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, ormore) listed in Table 2. In some such embodiments, one or more mutationsmay come from two or more variants as listed in Table 2. In someembodiments, an RNA comprises a nucleotide sequence encoding aSARS-CoV-2 S protein comprising each of the mutations identified inTable 2 as being characteristic of a certain Omicron variant (e.g., insome embodiments, an RNA comprises a nucleotide sequence encoding aSARS-CoV-2 S protein comprising each of the mutations listed in Table 2as being characteristic of an Omicron BA.1, BA.2, BA.2.12.1, BA.4/5,BA.2.75, BA.2.75.1, BA.4.6, BQ.1.1, XBB, XBB.1, XBB.2, or XBB.1.3variant).

In some embodiments, an RNA disclosed herein comprises a nucleotidesequence that encodes an immunogenic fragment of the SARS-Cov-2 Sprotein (e.g., the RBD) and which comprises one or more mutations thatare characteristic of a SARS-CoV-2 variant (e.g., an Omicron variantdescribed herein). For example, in some embodiments, an RNA comprises anucleotide sequence encoding the RBD of an S protein of a SARS-CoV-2variant (e.g., a region of the S protein corresponding to amino acids327 to 528 of SEQ ID NO: 1, and comprising one or more mutations thatare characteristic of a variant of concern that lie within this regionof the S protein).

In some embodiments, an RNA encodes a SARS-CoV-2 S protein comprising asubset of the mutations listed in Table 2. In some embodiments, an RNAencodes a SARS-CoV-2 S protein comprising the mutations listed in Table2 that are most prevalent in a certain variant (e.g., mutations thathave been detected in at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% of sequencescollected to date for a given variant sequenced). Mutation prevalencecan be determined, e.g., based on published sequences (e.g., sequencesthat are collected and made available to the public by GISAID).

In some embodiments, RNA described herein encodes a SARS-CoV-2 S proteincomprising one or more mutations that are characteristic of a BA.4/5variant. In some embodiments, the one or more mutations characteristicof a BA.4/5 variant include T19I, Δ24-26, A27S, Δ69/70, G142D, V213G,G339D, S371F, S373P, S375F, T376A, D405N, K417N, N440K, L452R, S477N,T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H,N764K, D796Y, Q954H, and N969K. In some embodiments, RNA describedherein encodes a SARS-CoV-2 S protein comprising one or more mutationsthat are characteristic of a BA.4/5 variant and excludes R408S. In someembodiments, RNA described herein encodes a SARS-CoV-2 S proteincomprising one or more mutations (including, e.g., 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) that arecharacteristic of a BA.4/5 variant and excludes R408S.

In some embodiments, RNA described herein encodes a SARS-CoV-2 S proteincomprising one or more (including, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, or more) mutations characteristic ofa BA.2.75 variant. In some embodiments, the one or more mutationscharacteristic of a BA.2.75 variant include T19I, Δ24-26, A27S, G142D,K147E, W152R, F157L, I210V, V213G, G257S, G339H, S371F, S373P, S375F,T376A, D405N, R408S, K417N, N440K, G446S, N460K, S477N, T478K, E484A,Q498R, N501Y, Y505H D614G, H655Y, N679K, P681H, N764K, Q954H, and N969K.In some embodiments, RNA described herein encodes a SARS-CoV-2 S proteincomprising one or more mutations (including, e.g., 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) that arecharacteristic of a BA.2.75 variant, and which excludes N354D. In someembodiments, RNA described herein encodes a SARS-CoV-2 S proteincomprising one or more mutations (including, e.g., 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) that arecharacteristic of a BA.2.75 variant, and which excludes D796Y. In someembodiments, RNA described herein encodes a SARS-CoV-2 S proteincomprising one or more mutations (including, e.g., 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) that arecharacteristic of a BA.2.75 variant, and which excludes D796Y and N354D.

In some embodiments, RNA described herein encodes a SARS-CoV-2 S proteincomprising one or more mutations characteristic of a BA.2.75.2 variant.In some embodiments, the one or more mutations characteristic of aBA.2.75.2 variant include T19I, Δ24-26, A27S, G142D, K147E, W152R,F157L, I210V, V213G, G257S, G339H, R346T, N354D, S371F, S373P, S375F,T376A, D405N, R408S, K417N, N440K, G446S, N460K, S477N, T478K, E484A,F4865, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y,Q954H, N969K, and D1199N. In some embodiments, RNA described hereinencodes a SARS-CoV-2 S protein comprising one or more mutations(including, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, or more) that are characteristic of a BA.2.75.2 variant,and which excludes R346T.

In some embodiments, RNA described herein encodes a SARS-CoV-2 S proteincomprising one or more mutations characteristic of a BA.4.6 or BF.7variant. In some embodiments, the one or more mutations characteristicof a BA.4.6 or BF.7 variant include T19I, Δ24-26, A27S, Δ69/70, G142D,V213G, G339D, R346T, 5371F, S373P, S375F, T376A, D405N, K417N, N440K,L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y,N679K, P681H, N764K, D796Y, Q954H, and N969K. In some embodiments, RNAdescribed herein encodes a SARS-CoV-2 S protein comprising one or moremutations (including, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, or more) that are characteristic of a BA.4.6 orBF.7 variant, and which exclude R408S. In some embodiments, RNAdescribed herein encodes a SARS-CoV-2 S protein comprising one or moremutations (including, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, or more) that are characteristic of a BA.4.6 orBF.7 variant, and which exclude N658S. In some embodiments, RNAdescribed herein encodes a SARS-CoV-2 S protein comprising one or moremutations (including, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, or more) that are characteristic of a BA.4.6 orBF.7 variant, and which exclude N658S and R408S.

In some embodiments, RNA described herein encodes a SARS-CoV-2 S proteincomprising one or more mutations characteristic of an Omicron XBBvariant. In some embodiments, the one or more mutations characteristicof an Omicron XBB variant include T19I, Δ24-26, A27S, V83A, G142D, A144,H146Q, Q183E, V213E, G339H, R346T, L368I, S371F, S373P, 5375F, T376A,D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A,F486S, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K,D796Y, Q954H, and N969K.

In some embodiments, RNA described herein encodes a SARS-CoV-2 S proteincomprising one or more mutations characteristic of an Omicron XBB.1variant. In some embodiments, the one or more mutations characteristicof an Omicron XBB.1 variant include G252V. In some embodiments, the oneor more mutations characteristic of an Omicron XBB.1 variant includeT19I, Δ24-26, A27S, V83A, G142D, A144, H146Q, Q183E, V213E, G252V,G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N,N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486S, F490S, Q498R,N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, andN969K. In some embodiments, RNA described herein encodes a SARS-CoV-2 Sprotein comprising one or more mutations (including, e.g., 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) thatare characteristic of an Omicron XBB.1 variant and which exclude Q493R.In some embodiments, RNA described herein encodes a SARS-CoV-2 S proteincomprising one or more mutations (including, e.g., 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) that arecharacteristic of an Omicron XBB variant and which exclude Q493R andG252V.

In some embodiments, RNA described herein encodes a SARS-CoV-2 S proteincomprising one or more mutations characteristic of an Omicron XBB.2variant. In some embodiments, the one or more mutations characteristicof an Omicron XBB.2 variant include D253G. In some embodiments, the oneor more mutations characteristic of an Omicron-XBB.2 variant includeT19I, Δ24-26, A27S, V83A, G142D, A144, H146Q Q183E, V213E, D253G, G339H,R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K,V445P, G446S, N460K, S477N, T478K, E484A, F486S, F490S, Q493R, Q498R,N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, andN969K.

In some embodiments, RNA described herein encodes a SARS-CoV-2 S proteincomprising one or more mutations characteristic of an Omicron XBB.1.3variant. In some embodiments, the one or more mutations characteristicof an Omicron XBB.1.3 variant include G252V and A484T. In someembodiments, the one or more mutations characteristic of an OmicronXBB.1.3 variant include T19I, Δ24-26, A27S, V83A, G142D, A144, H146,Q183E, V213E, G252V, G339H, R346T, 13681, S371F, S373P, S375F, T376A,D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, A484T,F486S, F490S, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H,N764K, D796Y, Q954H, and N969K.

In some embodiments, RNA described herein encodes a SARS-CoV-2 S proteincomprising one or more mutations that are characteristic of a BQ.1.1variant. In some embodiments, the one or more mutations characteristicof a BQ.1.1 variant include T19I, Δ24-26, A27S, Δ69/70, G142D, V213G,G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, K444T,L452R, N463K, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G,H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K. In someembodiments, RNA described herein encodes a SARS-CoV-2 S proteincomprising one or more mutations (including, e.g., 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) that arecharacteristic of a BQ.1.1 variant.

In one embodiment, the vaccine antigen described herein comprises,consists essentially of or consists of a spike protein (S) ofSARS-CoV-2, a variant thereof, or an immunogenic fragment thereof (e.g.,but not limited to RBD).

In one embodiment, a vaccine antigen comprises the amino acid sequenceof amino acids 17 to 1273 of SEQ ID NO: 1 or 7, an amino acid sequencehaving at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity tothe amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7,or an immunogenic fragment of the amino acid sequence of amino acids 17to 1273 of SEQ ID NO: 1 or 7, or the amino acid sequence having at least99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acidsequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7. In oneembodiment, a vaccine antigen comprises the amino acid sequence of aminoacids 17 to 1273 of SEQ ID NO: 1 or 7.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8 or 9, anucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the nucleotide sequence of nucleotides 49 to 3819 ofSEQ ID NO: 2, 8 or 9, or a fragment of the nucleotide sequence ofnucleotides 49 to 3819 of SEQ ID NO: 2, 8 or 9, or the nucleotidesequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the nucleotide sequence of nucleotides 49 to 3819 of SEQ IDNO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising theamino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7, anamino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the amino acid sequence of amino acids 17 to 1273 ofSEQ ID NO: 1 or 7, or an immunogenic fragment of the amino acid sequenceof amino acids 17 to 1273 of SEQ ID NO: 1 or 7, or the amino acidsequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the amino acid sequence of amino acids 17 to 1273 of SEQ IDNO: 1 or 7. In one embodiment, RNA encoding a vaccine antigen (i)comprises the nucleotide sequence of nucleotides 49 to 3819 of SEQ IDNO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising theamino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7.

In one embodiment, the vaccine antigen comprises, consists essentiallyof or consists of SARS-CoV-2 spike S1 fragment (S1) (the S1 subunit of aspike protein (S) of SARS-CoV-2), a variant thereof, or a fragmentthereof.

In one embodiment, a vaccine antigen comprises the amino acid sequenceof amino acids 17 to 683 of SEQ ID NO: 1, an amino acid sequence havingat least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 17 to 683 of SEQ ID NO: 1, or animmunogenic fragment of the amino acid sequence of amino acids 17 to 683of SEQ ID NO: 1, or the amino acid sequence having at least 99%, 98%,97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence ofamino acids 17 to 683 of SEQ ID NO: 1. In one embodiment, a vaccineantigen comprises the amino acid sequence of amino acids 17 to 683 ofSEQ ID NO: 1.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of nucleotides 49 to 2049 of SEQ ID NO: 2, 8 or 9, anucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the nucleotide sequence of nucleotides 49 to 2049 ofSEQ ID NO: 2, 8 or 9, or a fragment of the nucleotide sequence ofnucleotides 49 to 2049 of SEQ ID NO: 2, 8 or 9, or the nucleotidesequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the nucleotide sequence of nucleotides 49 to 2049 of SEQ IDNO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising theamino acid sequence of amino acids 17 to 683 of SEQ ID NO: 1, an aminoacid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the amino acid sequence of amino acids 17 to 683 of SEQ IDNO: 1, or an immunogenic fragment of the amino acid sequence of aminoacids 17 to 683 of SEQ ID NO: 1, or the amino acid sequence having atleast 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 17 to 683 of SEQ ID NO: 1. In oneembodiment, RNA encoding a vaccine antigen (i) comprises the nucleotidesequence of nucleotides 49 to 2049 of SEQ ID NO: 2, 8 or 9; and/or (ii)encodes an amino acid sequence comprising the amino acid sequence ofamino acids 17 to 683 of SEQ ID NO: 1.

In one embodiment, a vaccine antigen comprises the amino acid sequenceof amino acids 17 to 685 of SEQ ID NO: 1, an amino acid sequence havingat least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 17 to 685 of SEQ ID NO: 1, or animmunogenic fragment of the amino acid sequence of amino acids 17 to 685of SEQ ID NO: 1, or the amino acid sequence having at least 99%, 98%,97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence ofamino acids 17 to 685 of SEQ ID NO: 1. In one embodiment, a vaccineantigen comprises the amino acid sequence of amino acids 17 to 685 ofSEQ ID NO: 1.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of nucleotides 49 to 2055 of SEQ ID NO: 2, 8 or 9, anucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the nucleotide sequence of nucleotides 49 to 2055 ofSEQ ID NO: 2, 8 or 9, or a fragment of the nucleotide sequence ofnucleotides 49 to 2055 of SEQ ID NO: 2, 8 or 9, or the nucleotidesequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the nucleotide sequence of nucleotides 49 to 2055 of SEQ IDNO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising theamino acid sequence of amino acids 17 to 685 of SEQ ID NO: 1, an aminoacid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the amino acid sequence of amino acids 17 to 685 of SEQ IDNO: 1, or an immunogenic fragment of the amino acid sequence of aminoacids 17 to 685 of SEQ ID NO: 1, or the amino acid sequence having atleast 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 17 to 685 of SEQ ID NO: 1. In oneembodiment, RNA encoding a vaccine antigen (i) comprises the nucleotidesequence of nucleotides 49 to 2055 of SEQ ID NO: 2, 8 or 9; and/or (ii)encodes an amino acid sequence comprising the amino acid sequence ofamino acids 17 to 685 of SEQ ID NO: 1.

In one embodiment, a vaccine antigen comprises, consists essentially ofor consists of the receptor binding domain (RBD) of the S1 subunit of aspike protein (S) of SARS-CoV-2, a variant thereof, or a fragmentthereof. The amino acid sequence of amino acids 327 to 528 of SEQ ID NO:1, a variant thereof, or a fragment thereof is also referred to hereinas “RBD” or “RBD domain”.

In one embodiment, a vaccine antigen comprises the amino acid sequenceof amino acids 327 to 528 of SEQ ID NO: 1, an amino acid sequence havingat least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 327 to 528 of SEQ ID NO: 1, or animmunogenic fragment of the amino acid sequence of amino acids 327 to528 of SEQ ID NO: 1, or the amino acid sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequenceof amino acids 327 to 528 of SEQ ID NO: 1. In one embodiment, a vaccineantigen comprises the amino acid sequence of amino acids 327 to 528 ofSEQ ID NO: 1.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2, 8 or 9,a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the nucleotide sequence of nucleotides 979 to 1584 ofSEQ ID NO: 2, 8 or 9, or a fragment of the nucleotide sequence ofnucleotides 979 to 1584 of SEQ ID NO: 2, 8 or 9, or the nucleotidesequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the nucleotide sequence of nucleotides 979 to 1584 of SEQ IDNO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising theamino acid sequence of amino acids 327 to 528 of SEQ ID NO: 1, an aminoacid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the amino acid sequence of amino acids 327 to 528 of SEQ IDNO: 1, or an immunogenic fragment of the amino acid sequence of aminoacids 327 to 528 of SEQ ID NO: 1, or the amino acid sequence having atleast 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 327 to 528 of SEQ ID NO: 1. In oneembodiment, RNA encoding a vaccine antigen (i) comprises the nucleotidesequence of nucleotides 979 to 1584 of SEQ ID NO: 2, 8 or 9; and/or (ii)encodes an amino acid sequence comprising the amino acid sequence ofamino acids 327 to 528 of SEQ ID NO: 1.

According to certain embodiments, a signal peptide is fused, eitherdirectly or through a linker, to a SARS-CoV-2 S protein, a variantthereof, or a fragment thereof, i.e., the antigenic peptide or protein.Accordingly, in one embodiment, a signal peptide is fused to the abovedescribed amino acid sequences derived from SARS-CoV-2 S protein orimmunogenic fragments thereof (antigenic peptides or proteins) comprisedby the vaccine antigens described above.

Such signal peptides are sequences, which typically exhibit a length ofabout 15 to 30 amino acids and are preferably located at the N-terminusof the antigenic peptide or protein, without being limited thereto.Signal peptides as defined herein preferably allow the transport of theantigenic peptide or protein as encoded by the RNA into a definedcellular compartment, preferably the cell surface, the endoplasmicreticulum (ER) or the endosomal-lysosomal compartment. In oneembodiment, the signal peptide sequence as defined herein includes,without being limited thereto, the signal peptide sequence of SARS-CoV-2S protein, in particular a sequence comprising the amino acid sequenceof amino acids 1 to 16 or 1 to 19 of SEQ ID NO: 1 or a functionalvariant thereof.

In one embodiment, a signal sequence comprises the amino acid sequenceof amino acids 1 to 16 of SEQ ID NO: 1, an amino acid sequence having atleast 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 1 to 16 of SEQ ID NO: 1, or a functionalfragment of the amino acid sequence of amino acids 1 to 16 of SEQ ID NO:1, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%,90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to16 of SEQ ID NO: 1. In one embodiment, a signal sequence comprises theamino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1.

In one embodiment, RNA encoding a signal sequence (i) comprises thenucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8 or 9, anucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the nucleotide sequence of nucleotides 1 to 48 of SEQID NO: 2, 8 or 9, or a fragment of the nucleotide sequence ofnucleotides 1 to 48 of SEQ ID NO: 2, 8 or 9, or the nucleotide sequencehaving at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity tothe nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8 or 9;and/or (ii) encodes an amino acid sequence comprising the amino acidsequence of amino acids 1 to 16 of SEQ ID NO: 1, an amino acid sequencehaving at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity tothe amino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1, or afunctional fragment of the amino acid sequence of amino acids 1 to 16 ofSEQ ID NO: 1, or the amino acid sequence having at least 99%, 98%, 97%,96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of aminoacids 1 to 16 of SEQ ID NO: 1. In one embodiment, RNA encoding a signalsequence (i) comprises the nucleotide sequence of nucleotides 1 to 48 ofSEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequencecomprising the amino acid sequence of amino acids 1 to 16 of SEQ ID NO:1.

In one embodiment, a signal sequence comprises the amino acid sequenceof amino acids 1 to 19 of SEQ ID NO: 1, an amino acid sequence having atleast 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 1 to 19 of SEQ ID NO: 1, or a functionalfragment of the amino acid sequence of amino acids 1 to 19 of SEQ ID NO:1, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%,90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to19 of SEQ ID NO: 1. In one embodiment, a signal sequence comprises theamino acid sequence of amino acids 1 to 19 of SEQ ID NO: 1.

In one embodiment, RNA encoding a signal sequence (i) comprises thenucleotide sequence of nucleotides 1 to 57 of SEQ ID NO: 2, 8 or 9, anucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the nucleotide sequence of nucleotides 1 to 57 of SEQID NO: 2, 8 or 9, or a fragment of the nucleotide sequence ofnucleotides 1 to 57 of SEQ ID NO: 2, 8 or 9, or the nucleotide sequencehaving at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity tothe nucleotide sequence of nucleotides 1 to 57 of SEQ ID NO: 2, 8 or 9;and/or (ii) encodes an amino acid sequence comprising the amino acidsequence of amino acids 1 to 19 of SEQ ID NO: 1, an amino acid sequencehaving at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity tothe amino acid sequence of amino acids 1 to 19 of SEQ ID NO: 1, or afunctional fragment of the amino acid sequence of amino acids 1 to 19 ofSEQ ID NO: 1, or the amino acid sequence having at least 99%, 98%, 97%,96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of aminoacids 1 to 19 of SEQ ID NO: 1. In one embodiment, RNA encoding a signalsequence (i) comprises the nucleotide sequence of nucleotides 1 to 57 ofSEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequencecomprising the amino acid sequence of amino acids 1 to 19 of SEQ ID NO:1.

In some embodiments, a signal peptide sequence as defined hereinincludes, without being limited thereto, the signal peptide sequence ofan immunoglobulin, e.g., the signal peptide sequence of animmunoglobulin heavy chain variable region, wherein the immunoglobulinmay be human immunoglobulin. In particular, in some embodiments thesignal peptide sequence as defined herein includes a sequence comprisingthe amino acid sequence of amino acids 1 to 22 of SEQ ID NO: 31 or afunctional variant thereof.

In some embodiments, a signal peptide sequence is functional inmammalian cells.

In some embodiments, a utilized signal sequence is “intrinsic” in thatit is, in nature, it is associated with (e.g., linked to) the encodedpolypeptide.

In some embodiments, a utilized signal sequence is heterologous to theencoded polypeptide—e.g., is not naturally part of a polypeptide (e.g.,protein) whose sequences are included in the encoded polypeptide.

In some embodiments, signal peptides are sequences, which are typicallycharacterized by a length of about 15 to 30 amino acids.

In many embodiments, signal peptides are positioned at the N-terminus ofan encoded polypeptide as described herein, without being limitedthereto. In some embodiments, signal peptides preferably allow thetransport of the polypeptide encoded by RNAs of the present disclosurewith which they are associated into a defined cellular compartment,preferably the cell surface, the endoplasmic reticulum (ER) or theendosomal-lysosomal compartment.

In some embodiments, a signal sequence is selected from an S152 signalpeptide (aa 1-19), an immunoglobulin secretory signal peptide (aa 1-22),an HSV-1 gD signal peptide (MGGAAARLGAVILFVVIVGLHGVRSKY (SEQ ID NO:105)), an HSV-2 gD signal peptide (MGRLTSGVGTAALLVVAVGLRVVCA (SEQ ID NO:106)); a human SPARC signal peptide, a human insulin isoform 1 signalpeptide, a human albumin signal peptide, etc. Those skilled in the artwill be aware of other secretory signal peptides such as, for example,as disclosed in WO2017/081082 (e.g., SEQ ID NOs: 1-1115 and 1728, orfragments variants thereof) and WO2019008001.

In some embodiments, an RNA sequence encodes an epitope that maycomprise or otherwise be linked to a signal sequence (e.g., secretorysequence), such as those listed in Table A, or at least a sequencehaving 1, 2, 3, 4, or 5 amino acid differences relative thereto. In someembodiments, a signal sequence such as MFVFLVLLPLVSSQCVNLT (SEQ ID NO:108), or at least a sequence having 1, 2, 3, 4, or at the most 5 aminoacid differences relative thereto is utilized. In some embodiments, asequence such as MFVFLVLLPLVSSQCVNLT (SEQ ID NO: 108), or a sequencehaving 1, 2, 3, 4, or at the most 5 amino acid differences relativethereto, is utilized. In some embodiments, a signal sequence is selectedfrom those included in the Table A below and/or those encoded by thesequences in Table B below:

TABLE A Exemplary signal sequences SEQ ID NO: SignalSequence (Amino Acid) 105 HSV-1 gD SP MGGAAARLGAVILFVVIVGLHGVRS KY 106HSV-2 gD SP MGRLTSGVGTAALLVVAVGLRVVCA 107 HSV-2 MGRLTSGVGTAALLVVAVGLRVVCAKYA 108 SARS-COV-2-S MFVFLVLLPLVSSQCVNLT 109 human IgMDWIWRILFLVGAATGAHSQM heavy chain signal peptide (huSec) 110HuIgGk signal METPAQLLFLLLLWLPDTTG peptide 111 IgE heavyMDWTWILFLVAAATRVHS chain epsilon- 1signal peptide 112 JapaneseMLGSNSGQRVVFTILLLLVAPAYS encephalitis PRM signal sequence 113VSVg protein MKCLLYLAFLFIGVNCA signal sequence 114 MDWTWILFLVAAATRVHS115 ETPAQLLFLLLLWLPDTTG 116 MLGSNSGQRVVFTILLLLVAPAYS 117MKCLLYLAFLFIGVNCA 118 MWLVSLAIVTACAGA 119 MFVFLVLLPLVSSQC

TABLE B Exemplary nucleotide sequences encoding signal sequences SEQ IDNO: Signal Sequence (Nucleotide) 120 HSV-1 ATGGGGGGGGCTGCCGCCAGGTTGGGgD SP GGCCGTGATTTTGTTTGTCGTCATAG wild-type TGGGCCTCCATGGGGTCCGCAGCAAATAT 121 HSV-1 gD SP ATGggaggagccGCCGCCagactgggaGC optimizedCGTGatcctgttcgtggtgatcGTGgga nucleotide ctgCATggagtgagaAGCaagtacsequence 122 SARS- ATGTTTGTGTTTCTTGTGCTGCTGCCTCT COV-2-STGTGTCTTCTCAGTGTGTGAATTTGACA 123 human Ig ATGGATTGGATTTGGAGAATCCTGTTCCTCheavy GTGGGAGCCGCTACAGGAGCCCACTCCCAG chain ATG signal peptide (huSec)

In some embodiments, an RNA utilized as described herein encodes amultimerization element (e.g., a heterologous multimerization element).In some embodiments, a heterologous multimerization element comprises adimerization, trimerization or tetramerization element.

In some embodiments, a multimerization element is one described inWO2017/081082 (e.g., SEQ ID NOs: 1116-1167, or fragments or variantsthereof).

Exemplary trimerization and tetramerization elements include, but arenot limited to, engineered leucine zippers, fibritin foldon domain fromenterobacteria phage T4, GCN4pll, GCN4-pll, and p53.

In some embodiments, a provided encoded polypeptide(s) is able to form atrimeric complex. For example, a utilized encoded polypeptide(s) maycomprise a domain allowing formation of a multimeric complex, such asfor example particular a trimeric complex of an amino acid sequencecomprising an encoded polypeptide(s) as described herein. In someembodiments, a domain allowing formation of a multimeric complexcomprises a trimerization domain, for example, a trimerization domain asdescribed herein.

In some embodiments, an encoded polypeptide(s) can be modified byaddition of a T4-fibritin-derived “foldon” trimerization domain, forexample, to increase its immunogenicity.

In some embodiments, an RNAs described herein encodes a membraneassociation element (e.g., a heterologous membrane association element),such as a transmembrane domain.

A transmembrane domain can be N-terminal, C-terminal, or internal to anencoded polypeptide. A coding sequence of a transmembrane element istypically placed in frame (i.e., in the same reading frame), 5′, 3′, orinternal to coding sequences of sequences (e.g., sequences encodingpolypeptide(s)) with which it is to be linked.

In some embodiments, a transmembrane domain comprises or is atransmembrane domain of Hemagglutinin (HA) of Influenza virus, Env ofHIV-1, equine infectious anaemia virus (EIAV), murine leukaemia virus(MLV), mouse mammary tumor virus, G protein of vesicular stomatitisvirus (VSV), Rabies virus, or a seven transmembrane domain receptor.

In one embodiment, a signal sequence comprises the amino acid sequenceof amino acids 1 to 22 of SEQ ID NO: 31, an amino acid sequence havingat least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 1 to 22 of SEQ ID NO: 31, or a functionalfragment of the amino acid sequence of amino acids 1 to 22 of SEQ ID NO:31, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%,90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to22 of SEQ ID NO: 31. In one embodiment, a signal sequence comprises theamino acid sequence of amino acids 1 to 22 of SEQ ID NO: 31.

In one embodiment, RNA encoding a signal sequence (i) comprises thenucleotide sequence of nucleotides 54 to 119 of SEQ ID NO: 32, anucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the nucleotide sequence of nucleotides 54 to 119 ofSEQ ID NO: 32, or a fragment of the nucleotide sequence of nucleotides54 to 119 of SEQ ID NO: 32, or the nucleotide sequence having at least99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotidesequence of nucleotides 54 to 119 of SEQ ID NO: 32; and/or (ii) encodesan amino acid sequence comprising the amino acid sequence of amino acids1 to 22 of SEQ ID NO: 31, an amino acid sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequenceof amino acids 1 to 22 of SEQ ID NO: 31, or a functional fragment of theamino acid sequence of amino acids 1 to 22 of SEQ ID NO: 31, or theamino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the amino acid sequence of amino acids 1 to 22 of SEQID NO: 31. In one embodiment, RNA encoding a signal sequence (i)comprises the nucleotide sequence of nucleotides 54 to 119 of SEQ ID NO:32; and/or (ii) encodes an amino acid sequence comprising the amino acidsequence of amino acids 1 to 22 of SEQ ID NO: 31.

Such signal peptides are preferably used in order to promote secretionof the encoded antigenic peptide or protein. More preferably, a signalpeptide as defined herein is fused to an encoded antigenic peptide orprotein as defined herein.

Accordingly, in particularly preferred embodiments, the RNA describedherein comprises at least one coding region encoding an antigenicpeptide or protein and a signal peptide, said signal peptide preferablybeing fused to the antigenic peptide or protein, more preferably to theN-terminus of the antigenic peptide or protein as described herein.

In one embodiment, a vaccine antigen comprises the amino acid sequenceof SEQ ID NO: 1 or 7, an amino acid sequence having at least 99%, 98%,97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence ofSEQ ID NO: 1 or 7, or an immunogenic fragment of the amino acid sequenceof SEQ ID NO: 1 or 7, or the amino acid sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequenceof SEQ ID NO: 1 or 7. In one embodiment, a vaccine antigen comprises theamino acid sequence of SEQ ID NO: 1 or 7.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of SEQ ID NO: 2, 8 or 9, a nucleotide sequencehaving at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity tothe nucleotide sequence of SEQ ID NO: 2, 8 or 9, or a fragment of thenucleotide sequence of SEQ ID NO: 2, 8 or 9, or the nucleotide sequencehaving at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity tothe nucleotide sequence of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes anamino acid sequence comprising the amino acid sequence of SEQ ID NO: 1or 7, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%,90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 1 or7, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 1or 7, or the amino acid sequence having at least 99%, 98%, 97%, 96%,95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:1 or 7. In one embodiment, RNA encoding a vaccine antigen (i) comprisesthe nucleotide sequence of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes anamino acid sequence comprising the amino acid sequence of SEQ ID NO: 1or 7.

In one embodiment, a vaccine antigen comprises the amino acid sequenceof SEQ ID NO: 7, an amino acid sequence having at least 99%, 98%, 97%,96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ IDNO: 7, or an immunogenic fragment of the amino acid sequence of SEQ IDNO: 7, or the amino acid sequence having at least 99%, 98%, 97%, 96%,95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:7. In one embodiment, a vaccine antigen comprises the amino acidsequence of SEQ ID NO: 7.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of SEQ ID NO: 15, 16, 19, 20, 24, or 25, anucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the nucleotide sequence of SEQ ID NO: 15, 16, 19, 20,24, or 25, or a fragment of the nucleotide sequence of SEQ ID NO: 15,16, 19, 20, 24, or 25, or the nucleotide sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequenceof SEQ ID NO: 15, 16, 19, 20, 24, or 25; and/or (ii) encodes an aminoacid sequence comprising the amino acid sequence of SEQ ID NO: 7, anamino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the amino acid sequence of SEQ ID NO: 7, or animmunogenic fragment of the amino acid sequence of SEQ ID NO: 7, or theamino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the amino acid sequence of SEQ ID NO: 7. In oneembodiment, RNA encoding a vaccine antigen (i) comprises the nucleotidesequence of SEQ ID NO: 15, 16, 19, 20, 24, or 25; and/or (ii) encodes anamino acid sequence comprising the amino acid sequence of SEQ ID NO: 7.

In one embodiment, a vaccine antigen comprises the amino acid sequenceof amino acids 1 to 683 of SEQ ID NO: 1, an amino acid sequence havingat least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 1 to 683 of SEQ ID NO: 1, or an immunogenicfragment of the amino acid sequence of amino acids 1 to 683 of SEQ IDNO: 1, or the amino acid sequence having at least 99%, 98%, 97%, 96%,95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids1 to 683 of SEQ ID NO: 1. In one embodiment, a vaccine antigen comprisesthe amino acid sequence of amino acids 1 to 683 of SEQ ID NO: 1.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of nucleotides 1 to 2049 of SEQ ID NO: 2, 8 or 9, anucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the nucleotide sequence of nucleotides 1 to 2049 ofSEQ ID NO: 2, 8 or 9, or a fragment of the nucleotide sequence ofnucleotides 1 to 2049 of SEQ ID NO: 2, 8 or 9, or the nucleotidesequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the nucleotide sequence of nucleotides 1 to 2049 of SEQ IDNO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising theamino acid sequence of amino acids 1 to 683 of SEQ ID NO: 1, an aminoacid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the amino acid sequence of amino acids 1 to 683 of SEQ IDNO: 1, or an immunogenic fragment of the amino acid sequence of aminoacids 1 to 683 of SEQ ID NO: 1, or the amino acid sequence having atleast 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 1 to 683 of SEQ ID NO: 1. In oneembodiment, RNA encoding a vaccine antigen (i) comprises the nucleotidesequence of nucleotides 1 to 2049 of SEQ ID NO: 2, 8 or 9; and/or (ii)encodes an amino acid sequence comprising the amino acid sequence ofamino acids 1 to 683 of SEQ ID NO: 1.

In one embodiment, a vaccine antigen comprises the amino acid sequenceof amino acids 1 to 685 of SEQ ID NO: 1, an amino acid sequence havingat least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 1 to 685 of SEQ ID NO: 1, or an immunogenicfragment of the amino acid sequence of amino acids 1 to 685 of SEQ IDNO: 1, or the amino acid sequence having at least 99%, 98%, 97%, 96%,95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids1 to 685 of SEQ ID NO: 1. In one embodiment, a vaccine antigen comprisesthe amino acid sequence of amino acids 1 to 685 of SEQ ID NO: 1.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of nucleotides 1 to 2055 of SEQ ID NO: 2, 8 or 9, anucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the nucleotide sequence of nucleotides 1 to 2055 ofSEQ ID NO: 2, 8 or 9, or a fragment of the nucleotide sequence ofnucleotides 1 to 2055 of SEQ ID NO: 2, 8 or 9, or the nucleotidesequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the nucleotide sequence of nucleotides 1 to 2055 of SEQ IDNO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising theamino acid sequence of amino acids 1 to 685 of SEQ ID NO: 1, an aminoacid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the amino acid sequence of amino acids 1 to 685 of SEQ IDNO: 1, or an immunogenic fragment of the amino acid sequence of aminoacids 1 to 685 of SEQ ID NO: 1, or the amino acid sequence having atleast 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 1 to 685 of SEQ ID NO: 1. In oneembodiment, RNA encoding a vaccine antigen (i) comprises the nucleotidesequence of nucleotides 1 to 2055 of SEQ ID NO: 2, 8 or 9; and/or (ii)encodes an amino acid sequence comprising the amino acid sequence ofamino acids 1 to 685 of SEQ ID NO: 1.

In one embodiment, a vaccine antigen comprises the amino acid sequenceof SEQ ID NO: 3, an amino acid sequence having at least 99%, 98%, 97%,96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ IDNO: 3, or an immunogenic fragment of the amino acid sequence of SEQ IDNO: 3, or the amino acid sequence having at least 99%, 98%, 97%, 96%,95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:3. In one embodiment, a vaccine antigen comprises the amino acidsequence of SEQ ID NO: 3.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of SEQ ID NO: 4, a nucleotide sequence having atleast 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to thenucleotide sequence of SEQ ID NO: 4, or a fragment of the nucleotidesequence of SEQ ID NO: 4, or the nucleotide sequence having at least99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotidesequence of SEQ ID NO: 4; and/or (ii) encodes an amino acid sequencecomprising the amino acid sequence of SEQ ID NO: 3, an amino acidsequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the amino acid sequence of SEQ ID NO: 3, or an immunogenicfragment of the amino acid sequence of SEQ ID NO: 3, or the amino acidsequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the amino acid sequence of SEQ ID NO: 3. In one embodiment,RNA encoding a vaccine antigen (i) comprises the nucleotide sequence ofSEQ ID NO: 4; and/or (ii) encodes an amino acid sequence comprising theamino acid sequence of SEQ ID NO: 3.

In one embodiment, a vaccine antigen comprises the amino acid sequenceof amino acids 1 to 221 of SEQ ID NO: 29, an amino acid sequence havingat least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 1 to 221 of SEQ ID NO: 29, or animmunogenic fragment of the amino acid sequence of amino acids 1 to 221of SEQ ID NO: 29, or the amino acid sequence having at least 99%, 98%,97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence ofamino acids 1 to 221 of SEQ ID NO: 29. In one embodiment, a vaccineantigen comprises the amino acid sequence of amino acids 1 to 221 of SEQID NO: 29.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of nucleotides 54 to 716 of SEQ ID NO: 30, anucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the nucleotide sequence of nucleotides 54 to 716 ofSEQ ID NO: 30, or a fragment of the nucleotide sequence of nucleotides54 to 716 of SEQ ID NO: 30, or the nucleotide sequence having at least99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotidesequence of nucleotides 54 to 716 of SEQ ID NO: 30; and/or (ii) encodesan amino acid sequence comprising the amino acid sequence of amino acids1 to 221 of SEQ ID NO: 29, an amino acid sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequenceof amino acids 1 to 221 of SEQ ID NO: 29, or an immunogenic fragment ofthe amino acid sequence of amino acids 1 to 221 of SEQ ID NO: 29, or theamino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the amino acid sequence of amino acids 1 to 221 ofSEQ ID NO: 29. In one embodiment, RNA encoding a vaccine antigen (i)comprises the nucleotide sequence of nucleotides 54 to 716 of SEQ ID NO:30; and/or (ii) encodes an amino acid sequence comprising the amino acidsequence of amino acids 1 to 221 of SEQ ID NO: 29.

In one embodiment, a vaccine antigen comprises the amino acid sequenceof amino acids 1 to 224 of SEQ ID NO: 31, an amino acid sequence havingat least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 1 to 224 of SEQ ID NO: 31, or animmunogenic fragment of the amino acid sequence of amino acids 1 to 224of SEQ ID NO: 31, or the amino acid sequence having at least 99%, 98%,97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence ofamino acids 1 to 224 of SEQ ID NO: 31. In one embodiment, a vaccineantigen comprises the amino acid sequence of amino acids 1 to 224 of SEQID NO: 31.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of nucleotides 54 to 725 of SEQ ID NO: 32, anucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the nucleotide sequence of nucleotides 54 to 725 ofSEQ ID NO: 32, or a fragment of the nucleotide sequence of nucleotides54 to 725 of SEQ ID NO: 32, or the nucleotide sequence having at least99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotidesequence of nucleotides 54 to 725 of SEQ ID NO: 32; and/or (ii) encodesan amino acid sequence comprising the amino acid sequence of amino acids1 to 224 of SEQ ID NO: 31, an amino acid sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequenceof amino acids 1 to 224 of SEQ ID NO: 31, or an immunogenic fragment ofthe amino acid sequence of amino acids 1 to 224 of SEQ ID NO: 31, or theamino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the amino acid sequence of amino acids 1 to 224 ofSEQ ID NO: 31. In one embodiment, RNA encoding a vaccine antigen (i)comprises the nucleotide sequence of nucleotides 54 to 725 of SEQ ID NO:32; and/or (ii) encodes an amino acid sequence comprising the amino acidsequence of amino acids 1 to 224 of SEQ ID NO: 31.

According to certain embodiments, a trimerization domain is fused,either directly or through a linker, e.g., a glycine/serine linker, to aSARS-CoV-2 S protein, a variant thereof, or a fragment thereof, i.e.,the antigenic peptide or protein. Accordingly, in one embodiment, atrimerization domain is fused to the above described amino acidsequences derived from SARS-CoV-2 S protein or immunogenic fragmentsthereof (antigenic peptides or proteins) comprised by the vaccineantigens described above (which may optionally be fused to a signalpeptide as described above).

Such trimerization domains are preferably located at the C-terminus ofthe antigenic peptide or protein, without being limited thereto.Trimerization domains as defined herein preferably allow thetrimerization of the antigenic peptide or protein as encoded by the RNA.Examples of trimerization domains as defined herein include, withoutbeing limited thereto, foldon, the natural trimerization domain of T4fibritin. The C-terminal domain of T4 fibritin (foldon) is obligatoryfor the formation of the fibritin trimer structure and can be used as anartificial trimerization domain. In one embodiment, the trimerizationdomain as defined herein includes, without being limited thereto, asequence comprising the amino acid sequence of amino acids 3 to 29 ofSEQ ID NO: 10 or a functional variant thereof. In one embodiment, thetrimerization domain as defined herein includes, without being limitedthereto, a sequence comprising the amino acid sequence of SEQ ID NO: 10or a functional variant thereof.

In one embodiment, a trimerization domain comprises the amino acidsequence of amino acids 3 to 29 of SEQ ID NO: 10, an amino acid sequencehaving at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity tothe amino acid sequence of amino acids 3 to 29 of SEQ ID NO: 10, or afunctional fragment of the amino acid sequence of amino acids 3 to 29 ofSEQ ID NO: 10, or the amino acid sequence having at least 99%, 98%, 97%,96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of aminoacids 3 to 29 of SEQ ID NO: 10. In one embodiment, a trimerizationdomain comprises the amino acid sequence of amino acids 3 to 29 of SEQID NO: 10.

In one embodiment, RNA encoding a trimerization domain (i) comprises thenucleotide sequence of nucleotides 7 to 87 of SEQ ID NO: 11, anucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the nucleotide sequence of nucleotides 7 to 87 of SEQID NO: 11, or a fragment of the nucleotide sequence of nucleotides 7 to87 of SEQ ID NO: 11, or the nucleotide sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequenceof nucleotides 7 to 87 of SEQ ID NO: 11; and/or (ii) encodes an aminoacid sequence comprising the amino acid sequence of amino acids 3 to 29of SEQ ID NO: 10, an amino acid sequence having at least 99%, 98%, 97%,96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of aminoacids 3 to 29 of SEQ ID NO: 10, or a functional fragment of the aminoacid sequence of amino acids 3 to 29 of SEQ ID NO: 10, or the amino acidsequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the amino acid sequence of amino acids 3 to 29 of SEQ ID NO:10. In one embodiment, RNA encoding a trimerization domain (i) comprisesthe nucleotide sequence of nucleotides 7 to 87 of SEQ ID NO: 11; and/or(ii) encodes an amino acid sequence comprising the amino acid sequenceof amino acids 3 to 29 of SEQ ID NO: 10.

In one embodiment, a trimerization domain comprises the amino acidsequence SEQ ID NO: 10, an amino acid sequence having at least 99%, 98%,97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence ofSEQ ID NO: 10, or a functional fragment of the amino acid sequence ofSEQ ID NO: 10, or the amino acid sequence having at least 99%, 98%, 97%,96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ IDNO: 10. In one embodiment, a trimerization domain comprises the aminoacid sequence of SEQ ID NO: 10. In one embodiment, RNA encoding atrimerization domain (i) comprises the nucleotide sequence of SEQ ID NO:11, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%,85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 11, or afragment of the nucleotide sequence of SEQ ID NO: 11, or the nucleotidesequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the nucleotide sequence of SEQ ID NO: 11; and/or (ii)encodes an amino acid sequence comprising the amino acid sequence of SEQID NO: 10, an amino acid sequence having at least 99%, 98%, 97%, 96%,95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:10, or a functional fragment of the amino acid sequence of SEQ ID NO:10, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%,90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 10.In one embodiment, RNA encoding a trimerization domain (i) comprises thenucleotide sequence of SEQ ID NO: 11; and/or (ii) encodes an amino acidsequence comprising the amino acid sequence of SEQ ID NO: 10.

Such trimerization domains are preferably used in order to promotetrimerization of the encoded antigenic peptide or protein. Morepreferably, a trimerization domain as defined herein is fused to anantigenic peptide or protein as defined herein.

Accordingly, in particularly preferred embodiments, the RNA describedherein comprises at least one coding region encoding an antigenicpeptide or protein and a trimerization domain as defined herein, saidtrimerization domain preferably being fused to the antigenic peptide orprotein, more preferably to the C-terminus of the antigenic peptide orprotein.

In one embodiment, a vaccine antigen comprises the amino acid sequenceof SEQ ID NO: 5, an amino acid sequence having at least 99%, 98%, 97%,96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ IDNO: 5, or an immunogenic fragment of the amino acid sequence of SEQ IDNO: 5, or the amino acid sequence having at least 99%, 98%, 97%, 96%,95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:S. In one embodiment, a vaccine antigen comprises the amino acidsequence of SEQ ID NO: 5.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of SEQ ID NO: 6, a nucleotide sequence having atleast 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to thenucleotide sequence of SEQ ID NO: 6, or a fragment of the nucleotidesequence of SEQ ID NO: 6, or the nucleotide sequence having at least99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotidesequence of SEQ ID NO: 6; and/or (ii) encodes an amino acid sequencecomprising the amino acid sequence of SEQ ID NO: 5, an amino acidsequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the amino acid sequence of SEQ ID NO: 5, or an immunogenicfragment of the amino acid sequence of SEQ ID NO: 5, or the amino acidsequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the amino acid sequence of SEQ ID NO: 5. In one embodiment,RNA encoding a vaccine antigen (i) comprises the nucleotide sequence ofSEQ ID NO: 6; and/or (ii) encodes an amino acid sequence comprising theamino acid sequence of SEQ ID NO: 5.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of SEQ ID NO: 17, 21, or 26, a nucleotide sequencehaving at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity tothe nucleotide sequence of SEQ ID NO: 17, 21, or 26, or a fragment ofthe nucleotide sequence of SEQ ID NO: 17, 21, or 26, or the nucleotidesequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the nucleotide sequence of SEQ ID NO: 17, 21, or 26; and/or(ii) encodes an amino acid sequence comprising the amino acid sequenceof SEQ ID NO: 5, an amino acid sequence having at least 99%, 98%, 97%,96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ IDNO: 5, or an immunogenic fragment of the amino acid sequence of SEQ IDNO: 5, or the amino acid sequence having at least 99%, 98%, 97%, 96%,95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:5. In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of SEQ ID NO: 17, 21, or 26; and/or (ii) encodes anamino acid sequence comprising the amino acid sequence of SEQ ID NO: 5.

In one embodiment, a vaccine antigen comprises the amino acid sequenceof SEQ ID NO: 18, an amino acid sequence having at least 99%, 98%, 97%,96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ IDNO: 18, or an immunogenic fragment of the amino acid sequence of SEQ IDNO: 18, or the amino acid sequence having at least 99%, 98%, 97%, 96%,95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:18. In one embodiment, a vaccine antigen comprises the amino acidsequence of SEQ ID NO: 18.

In one embodiment, a vaccine antigen comprises the amino acid sequenceof amino acids 1 to 257 of SEQ ID NO: 29, an amino acid sequence havingat least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 1 to 257 of SEQ ID NO: 29, or animmunogenic fragment of the amino acid sequence of amino acids 1 to 257of SEQ ID NO: 29, or the amino acid sequence having at least 99%, 98%,97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence ofamino acids 1 to 257 of SEQ ID NO: 29. In one embodiment, a vaccineantigen comprises the amino acid sequence of amino acids 1 to 257 of SEQID NO: 29.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of nucleotides 54 to 824 of SEQ ID NO: 30, anucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the nucleotide sequence of nucleotides 54 to 824 ofSEQ ID NO: 30, or a fragment of the nucleotide sequence of nucleotides54 to 824 of SEQ ID NO: 30, or the nucleotide sequence having at least99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotidesequence of nucleotides 54 to 824 of SEQ ID NO: 30; and/or (ii) encodesan amino acid sequence comprising the amino acid sequence of amino acids1 to 257 of SEQ ID NO: 29, an amino acid sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequenceof amino acids 1 to 257 of SEQ ID NO: 29, or an immunogenic fragment ofthe amino acid sequence of amino acids 1 to 257 of SEQ ID NO: 29, or theamino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the amino acid sequence of amino acids 1 to 257 ofSEQ ID NO: 29. In one embodiment, RNA encoding a vaccine antigen (i)comprises the nucleotide sequence of nucleotides 54 to 824 of SEQ ID NO:30; and/or (ii) encodes an amino acid sequence comprising the amino acidsequence of amino acids 1 to 257 of SEQ ID NO: 29.

In one embodiment, a vaccine antigen comprises the amino acid sequenceof amino acids 1 to 260 of SEQ ID NO: 31, an amino acid sequence havingat least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 1 to 260 of SEQ ID NO: 31, or animmunogenic fragment of the amino acid sequence of amino acids 1 to 260of SEQ ID NO: 31, or the amino acid sequence having at least 99%, 98%,97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence ofamino acids 1 to 260 of SEQ ID NO: 31. In one embodiment, a vaccineantigen comprises the amino acid sequence of amino acids 1 to 260 of SEQID NO: 31.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of nucleotides 54 to 833 of SEQ ID NO: 32, anucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the nucleotide sequence of nucleotides 54 to 833 ofSEQ ID NO: 32, or a fragment of the nucleotide sequence of nucleotides54 to 833 of SEQ ID NO: 32, or the nucleotide sequence having at least99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotidesequence of nucleotides 54 to 833 of SEQ ID NO: 32; and/or (ii) encodesan amino acid sequence comprising the amino acid sequence of amino acids1 to 260 of SEQ ID NO: 31, an amino acid sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequenceof amino acids 1 to 260 of SEQ ID NO: 31, or an immunogenic fragment ofthe amino acid sequence of amino acids 1 to 260 of SEQ ID NO: 31, or theamino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the amino acid sequence of amino acids 1 to 260 ofSEQ ID NO: 31. In one embodiment, RNA encoding a vaccine antigen (i)comprises the nucleotide sequence of nucleotides 54 to 833 of SEQ ID NO:32; and/or (ii) encodes an amino acid sequence comprising the amino acidsequence of amino acids 1 to 260 of SEQ ID NO: 31.

In one embodiment, a vaccine antigen comprises the amino acid sequenceof amino acids 20 to 257 of SEQ ID NO: 29, an amino acid sequence havingat least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 20 to 257 of SEQ ID NO: 29, or animmunogenic fragment of the amino acid sequence of amino acids 20 to 257of SEQ ID NO: 29, or the amino acid sequence having at least 99%, 98%,97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence ofamino acids 20 to 257 of SEQ ID NO: 29. In one embodiment, a vaccineantigen comprises the amino acid sequence of amino acids 20 to 257 ofSEQ ID NO: 29.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of nucleotides 111 to 824 of SEQ ID NO: 30, anucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the nucleotide sequence of nucleotides 111 to 824 ofSEQ ID NO: 30, or a fragment of the nucleotide sequence of nucleotides111 to 824 of SEQ ID NO: 30, or the nucleotide sequence having at least99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotidesequence of nucleotides 111 to 824 of SEQ ID NO: 30; and/or (ii) encodesan amino acid sequence comprising the amino acid sequence of amino acids20 to 257 of SEQ ID NO: 29, an amino acid sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequenceof amino acids 20 to 257 of SEQ ID NO: 29, or an immunogenic fragment ofthe amino acid sequence of amino acids 20 to 257 of SEQ ID NO: 29, orthe amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%,85%, or 80% identity to the amino acid sequence of amino acids 20 to 257of SEQ ID NO: 29. In one embodiment, RNA encoding a vaccine antigen (i)comprises the nucleotide sequence of nucleotides 111 to 824 of SEQ IDNO: 30; and/or (ii) encodes an amino acid sequence comprising the aminoacid sequence of amino acids 20 to 257 of SEQ ID NO: 29.

In one embodiment, a vaccine antigen comprises the amino acid sequenceof amino acids 23 to 260 of SEQ ID NO: 31, an amino acid sequence havingat least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 23 to 260 of SEQ ID NO: 31, or animmunogenic fragment of the amino acid sequence of amino acids 23 to 260of SEQ ID NO: 31, or the amino acid sequence having at least 99%, 98%,97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence ofamino acids 23 to 260 of SEQ ID NO: 31. In one embodiment, a vaccineantigen comprises the amino acid sequence of amino acids 23 to 260 ofSEQ ID NO: 31.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of nucleotides 120 to 833 of SEQ ID NO: 32, anucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the nucleotide sequence of nucleotides 120 to 833 ofSEQ ID NO: 32, or a fragment of the nucleotide sequence of nucleotides120 to 833 of SEQ ID NO: 32, or the nucleotide sequence having at least99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotidesequence of nucleotides 120 to 833 of SEQ ID NO: 32; and/or (ii) encodesan amino acid sequence comprising the amino acid sequence of amino acids23 to 260 of SEQ ID NO: 31, an amino acid sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequenceof amino acids 23 to 260 of SEQ ID NO: 31, or an immunogenic fragment ofthe amino acid sequence of amino acids 23 to 260 of SEQ ID NO: 31, orthe amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%,85%, or 80% identity to the amino acid sequence of amino acids 23 to 260of SEQ ID NO: 31. In one embodiment, RNA encoding a vaccine antigen (i)comprises the nucleotide sequence of nucleotides 120 to 833 of SEQ IDNO: 32; and/or (ii) encodes an amino acid sequence comprising the aminoacid sequence of amino acids 23 to 260 of SEQ ID NO: 31.

According to certain embodiments, a transmembrane domain is fused,either directly or through a linker, e.g., a glycine/serine linker, to aSARS-CoV-2 S protein, a variant thereof, or a fragment thereof, i.e.,the antigenic peptide or protein. Accordingly, in one embodiment, atransmembrane domain is fused to the above described amino acidsequences derived from SARS-CoV-2 S protein or immunogenic fragmentsthereof (antigenic peptides or proteins) comprised by the vaccineantigens described above (which may optionally be fused to a signalpeptide and/or trimerization domain as described above).

Such transmembrane domains are preferably located at the C-terminus ofthe antigenic peptide or protein, without being limited thereto.Preferably, such transmembrane domains are located at the C-terminus ofthe trimerization domain, if present, without being limited thereto. Inone embodiment, a trimerization domain is present between the SARS-CoV-2S protein, a variant thereof, or a fragment thereof, i.e., the antigenicpeptide or protein, and the transmembrane domain.

Transmembrane domains as defined herein preferably allow the anchoringinto a cellular membrane of the antigenic peptide or protein as encodedby the RNA.

In one embodiment, the transmembrane domain sequence as defined hereinincludes, without being limited thereto, the transmembrane domainsequence of SARS-CoV-2 S protein, in particular a sequence comprisingthe amino acid sequence of amino acids 1207 to 1254 of SEQ ID NO: 1 or afunctional variant thereof.

In one embodiment, a transmembrane domain sequence comprises the aminoacid sequence of amino acids 1207 to 1254 of SEQ ID NO: 1, an amino acidsequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the amino acid sequence of amino acids 1207 to 1254 of SEQID NO: 1, or a functional fragment of the amino acid sequence of aminoacids 1207 to 1254 of SEQ ID NO: 1, or the amino acid sequence having atleast 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 1207 to 1254 of SEQ ID NO: 1. In oneembodiment, a transmembrane domain sequence comprises the amino acidsequence of amino acids 1207 to 1254 of SEQ ID NO: 1.

In one embodiment, RNA encoding a transmembrane domain sequence (i)comprises the nucleotide sequence of nucleotides 3619 to 3762 of SEQ IDNO: 2, 8 or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%,95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides3619 to 3762 of SEQ ID NO: 2, 8 or 9, or a fragment of the nucleotidesequence of nucleotides 3619 to 3762 of SEQ ID NO: 2, 8 or 9, or thenucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the nucleotide sequence of nucleotides 3619 to 3762of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequencecomprising the amino acid sequence of amino acids 1207 to 1254 of SEQ IDNO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%,90%, 85%, or 80% identity to the amino acid sequence of amino acids 1207to 1254 of SEQ ID NO: 1, or a functional fragment of the amino acidsequence of amino acids 1207 to 1254 of SEQ ID NO: 1, or the amino acidsequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the amino acid sequence of amino acids 1207 to 1254 of SEQID NO: 1. In one embodiment, RNA encoding a transmembrane domainsequence (i) comprises the nucleotide sequence of nucleotides 3619 to3762 of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequencecomprising the amino acid sequence of amino acids 1207 to 1254 of SEQ IDNO: 1.

In one embodiment, a vaccine antigen comprises the amino acid sequenceof amino acids 1 to 311 of SEQ ID NO: 29, an amino acid sequence havingat least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 1 to 311 of SEQ ID NO: 29, or animmunogenic fragment of the amino acid sequence of amino acids 1 to 311of SEQ ID NO: 29, or the amino acid sequence having at least 99%, 98%,97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence ofamino acids 1 to 311 of SEQ ID NO: 29. In one embodiment, a vaccineantigen comprises the amino acid sequence of amino acids 1 to 311 of SEQID NO: 29.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of nucleotides 54 to 986 of SEQ ID NO: 30, anucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the nucleotide sequence of nucleotides 54 to 986 ofSEQ ID NO: 30, or a fragment of the nucleotide sequence of nucleotides54 to 986 of SEQ ID NO: 30, or the nucleotide sequence having at least99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotidesequence of nucleotides 54 to 986 of SEQ ID NO: 30; and/or (ii) encodesan amino acid sequence comprising the amino acid sequence of amino acids1 to 311 of SEQ ID NO: 29, an amino acid sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequenceof amino acids 1 to 311 of SEQ ID NO: 29, or an immunogenic fragment ofthe amino acid sequence of amino acids 1 to 311 of SEQ ID NO: 29, or theamino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the amino acid sequence of amino acids 1 to 311 ofSEQ ID NO: 29. In one embodiment, RNA encoding a vaccine antigen (i)comprises the nucleotide sequence of nucleotides 54 to 986 of SEQ ID NO:30; and/or (ii) encodes an amino acid sequence comprising the amino acidsequence of amino acids 1 to 311 of SEQ ID NO: 29.

In one embodiment, a vaccine antigen comprises the amino acid sequenceof amino acids 1 to 314 of SEQ ID NO: 31, an amino acid sequence havingat least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 1 to 314 of SEQ ID NO: 31, or animmunogenic fragment of the amino acid sequence of amino acids 1 to 314of SEQ ID NO: 31, or the amino acid sequence having at least 99%, 98%,97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence ofamino acids 1 to 314 of SEQ ID NO: 31. In one embodiment, a vaccineantigen comprises the amino acid sequence of amino acids 1 to 314 of SEQID NO: 31.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of nucleotides 54 to 995 of SEQ ID NO: 32, anucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the nucleotide sequence of nucleotides 54 to 995 ofSEQ ID NO: 32, or a fragment of the nucleotide sequence of nucleotides54 to 995 of SEQ ID NO: 32, or the nucleotide sequence having at least99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotidesequence of nucleotides 54 to 995 of SEQ ID NO: 32; and/or (ii) encodesan amino acid sequence comprising the amino acid sequence of amino acids1 to 314 of SEQ ID NO: 31, an amino acid sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequenceof amino acids 1 to 314 of SEQ ID NO: 31, or an immunogenic fragment ofthe amino acid sequence of amino acids 1 to 314 of SEQ ID NO: 31, or theamino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the amino acid sequence of amino acids 1 to 314 ofSEQ ID NO: 31. In one embodiment, RNA encoding a vaccine antigen (i)comprises the nucleotide sequence of nucleotides 54 to 995 of SEQ ID NO:32; and/or (ii) encodes an amino acid sequence comprising the amino acidsequence of amino acids 1 to 314 of SEQ ID NO: 31.

In one embodiment, a vaccine antigen comprises the amino acid sequenceof amino acids 20 to 311 of SEQ ID NO: 29, an amino acid sequence havingat least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 20 to 311 of SEQ ID NO: 29, or animmunogenic fragment of the amino acid sequence of amino acids 20 to 311of SEQ ID NO: 29, or the amino acid sequence having at least 99%, 98%,97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence ofamino acids 20 to 311 of SEQ ID NO: 29. In one embodiment, a vaccineantigen comprises the amino acid sequence of amino acids 20 to 311 ofSEQ ID NO: 29.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of nucleotides 111 to 986 of SEQ ID NO: 30, anucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the nucleotide sequence of nucleotides 111 to 986 ofSEQ ID NO: 30, or a fragment of the nucleotide sequence of nucleotides111 to 986 of SEQ ID NO: 30, or the nucleotide sequence having at least99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotidesequence of nucleotides 111 to 986 of SEQ ID NO: 30; and/or (ii) encodesan amino acid sequence comprising the amino acid sequence of amino acids20 to 311 of SEQ ID NO: 29, an amino acid sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequenceof amino acids 20 to 311 of SEQ ID NO: 29, or an immunogenic fragment ofthe amino acid sequence of amino acids 20 to 311 of SEQ ID NO: 29, orthe amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%,85%, or 80% identity to the amino acid sequence of amino acids 20 to 311of SEQ ID NO: 29. In one embodiment, RNA encoding a vaccine antigen (i)comprises the nucleotide sequence of nucleotides 111 to 986 of SEQ IDNO: 30; and/or (ii) encodes an amino acid sequence comprising the aminoacid sequence of amino acids 20 to 311 of SEQ ID NO: 29.

In one embodiment, a vaccine antigen comprises the amino acid sequenceof amino acids 23 to 314 of SEQ ID NO: 31, an amino acid sequence havingat least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of amino acids 23 to 314 of SEQ ID NO: 31, or animmunogenic fragment of the amino acid sequence of amino acids 23 to 314of SEQ ID NO: 31, or the amino acid sequence having at least 99%, 98%,97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence ofamino acids 23 to 314 of SEQ ID NO: 31. In one embodiment, a vaccineantigen comprises the amino acid sequence of amino acids 23 to 314 ofSEQ ID NO: 31.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of nucleotides 120 to 995 of SEQ ID NO: 32, anucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,or 80% identity to the nucleotide sequence of nucleotides 120 to 995 ofSEQ ID NO: 32, or a fragment of the nucleotide sequence of nucleotides120 to 995 of SEQ ID NO: 32, or the nucleotide sequence having at least99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotidesequence of nucleotides 120 to 995 of SEQ ID NO: 32; and/or (ii) encodesan amino acid sequence comprising the amino acid sequence of amino acids23 to 314 of SEQ ID NO: 31, an amino acid sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequenceof amino acids 23 to 314 of SEQ ID NO: 31, or an immunogenic fragment ofthe amino acid sequence of amino acids 23 to 314 of SEQ ID NO: 31, orthe amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%,85%, or 80% identity to the amino acid sequence of amino acids 23 to 314of SEQ ID NO: 31. In one embodiment, RNA encoding a vaccine antigen (i)comprises the nucleotide sequence of nucleotides 120 to 995 of SEQ IDNO: 32; and/or (ii) encodes an amino acid sequence comprising the aminoacid sequence of amino acids 23 to 314 of SEQ ID NO: 31.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of SEQ ID NO: 30, a nucleotide sequence having atleast 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to thenucleotide sequence of SEQ ID NO: 30, or a fragment of the nucleotidesequence of SEQ ID NO: 30, or the nucleotide sequence having at least99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotidesequence of SEQ ID NO: 30; and/or (ii) encodes an amino acid sequencecomprising the amino acid sequence of SEQ ID NO: 29, an amino acidsequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the amino acid sequence of SEQ ID NO: 29, or an immunogenicfragment of the amino acid sequence of SEQ ID NO: 29, or the amino acidsequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the amino acid sequence of SEQ ID NO: 29. In one embodiment,RNA encoding a vaccine antigen (i) comprises the nucleotide sequence ofSEQ ID NO: 30; and/or (ii) encodes an amino acid sequence comprising theamino acid sequence of SEQ ID NO: 29.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of SEQ ID NO: 32, a nucleotide sequence having atleast 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to thenucleotide sequence of SEQ ID NO: 32, or a fragment of the nucleotidesequence of SEQ ID NO: 32, or the nucleotide sequence having at least99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotidesequence of SEQ ID NO: 32; and/or (ii) encodes an amino acid sequencecomprising the amino acid sequence of SEQ ID NO: 31, an amino acidsequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the amino acid sequence of SEQ ID NO: 31, or an immunogenicfragment of the amino acid sequence of SEQ ID NO: 31, or the amino acidsequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the amino acid sequence of SEQ ID NO: 31. In one embodiment,RNA encoding a vaccine antigen (i) comprises the nucleotide sequence ofSEQ ID NO: 32; and/or (ii) encodes an amino acid sequence comprising theamino acid sequence of SEQ ID NO: 31.

In one embodiment, a vaccine antigen comprises the amino acid sequenceof SEQ ID NO: 28, an amino acid sequence having at least 99%, 98%, 97%,96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ IDNO: 28, or an immunogenic fragment of the amino acid sequence of SEQ IDNO: 28, or the amino acid sequence having at least 99%, 98%, 97%, 96%,95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:28. In one embodiment, a vaccine antigen comprises the amino acidsequence of SEQ ID NO: 28.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of SEQ ID NO: 27, a nucleotide sequence having atleast 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to thenucleotide sequence of SEQ ID NO: 27, or a fragment of the nucleotidesequence of SEQ ID NO: 27, or the nucleotide sequence having at least99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotidesequence of SEQ ID NO: 27; and/or (ii) encodes an amino acid sequencecomprising the amino acid sequence of SEQ ID NO: 28, an amino acidsequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the amino acid sequence of SEQ ID NO: 28, or an immunogenicfragment of the amino acid sequence of SEQ ID NO: 28, or the amino acidsequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the amino acid sequence of SEQ ID NO: 28. In one embodiment,RNA encoding a vaccine antigen (i) comprises the nucleotide sequence ofSEQ ID NO: 27; and/or (ii) encodes an amino acid sequence comprising theamino acid sequence of SEQ ID NO: 28.

In one embodiment, a vaccine antigen comprises the amino acid sequenceof SEQ ID NO: 49, an amino acid sequence having at least 99.5%, 99%,98.5%, 98%, 98.5% or 97% identity to the amino acid sequence of SEQ IDNO: 49, or an immunogenic fragment of the amino acid sequence of SEQ IDNO: 49, or the amino acid sequence having at least 99.5%, 99%, 98.5%,98%, 98.5% or 97% identity to the amino acid sequence of SEQ ID NO: 49.In one embodiment, a vaccine antigen comprises the amino acid sequenceof SEQ ID NO: 49. The amino acid sequence of SEQ ID NO: 49 correspondsto the amino acid sequence of the full-length S protein from OmicronBA.1, which includes proline residues at positions corresponding toresidues 986 and 987 of SEQ ID NO: 1 (residues 983 and 984 of SEQ ID NO:49).

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of SEQ ID NO: 50, a nucleotide sequence having atleast 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotidesequence of SEQ ID NO: 50, or a fragment of the nucleotide sequence ofSEQ ID NO: 50, or the nucleotide sequence having at least 99.5%, 99%,98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of SEQ IDNO: 50; and/or (ii) encodes an amino acid sequence comprising the aminoacid sequence of SEQ ID NO: 49, an amino acid sequence having at least99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequenceof SEQ ID NO: 49, or an immunogenic fragment of the amino acid sequenceof SEQ ID NO: 49, or the amino acid sequence having at least 99.5%, 99%,98.5%, 98%, 98.5% or 97% identity to the amino acid sequence of SEQ IDNO: 49. In one embodiment, RNA encoding a vaccine antigen (i) comprisesthe nucleotide sequence of SEQ ID NO: 50; and/or (ii) encodes an aminoacid sequence comprising the amino acid sequence of SEQ ID NO: 49. Thenucleotide sequence of SEQ ID NO: 50 is a nucleotide sequence designedto encode the amino acid sequence of the full-length S protein fromOmicron BA.1 with proline residues at positions corresponding toresidues 986 and 987 of SEQ ID NO: 1 (residues 983 and 984 of SEQ ID NO:49).

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of SEQ ID NO: 51, a nucleotide sequence having atleast 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotidesequence of SEQ ID NO: 51, or a fragment of the nucleotide sequence ofSEQ ID NO: 51, or the nucleotide sequence having at least 99.5%, 99%,98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of SEQ IDNO: 51; and/or (ii) encodes an amino acid sequence comprising the aminoacid sequence of SEQ ID NO: 49, an amino acid sequence having at least99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequenceof SEQ ID NO: 49, or an immunogenic fragment of the amino acid sequenceof SEQ ID NO: 49, or the amino acid sequence having at least 99.5%, 99%,98.5%, 98%, 98.5% or 97% identity to the amino acid sequence of SEQ IDNO: 49. In one embodiment, RNA encoding a vaccine antigen (i) comprisesthe nucleotide sequence of SEQ ID NO: 51; and/or (ii) encodes an aminoacid sequence comprising the amino acid sequence of SEQ ID NO: 49. Thenucleotide sequence of SEQ ID NO: 51 corresponds to an RNA construct(e.g., comprising a 5′ UTR, a S-protein-encoding sequence, a 3′ UTR, anda poly-A tail), which encodes the amino acid sequence of the full-lengthS protein from Omicron BA.1 with proline residues at positionscorresponding to residues 986 and 987 of SEQ ID NO: 1 (corresponding toresidues 983 and 984 of SEQ ID NO: 49).

In one embodiment, a vaccine antigen comprises the amino acid sequenceof SEQ ID NO: 55, an amino acid sequence having at least 99.5%, 99%,98.5%, 98%, 98.5% or 97% identity to the amino acid sequence of SEQ IDNO: 55, or an immunogenic fragment of the amino acid sequence of SEQ IDNO: 55, or the amino acid sequence having at least 99.5%, 99%, 98.5%,98%, 98.5% or 97% identity to the amino acid sequence of SEQ ID NO: 55.In one embodiment, a vaccine antigen comprises the amino acid sequenceof SEQ ID NO: 55.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of SEQ ID NO: 56, a nucleotide sequence having atleast 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotidesequence of SEQ ID NO: 56, or a fragment of the nucleotide sequence ofSEQ ID NO: 56, or the nucleotide sequence having at least 99.5%, 99%,98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of SEQ IDNO: 56; and/or (ii) encodes an amino acid sequence comprising the aminoacid sequence of SEQ ID NO: 55, an amino acid sequence having at least99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequenceof SEQ ID NO: 55, or an immunogenic fragment of the amino acid sequenceof SEQ ID NO: 55, or the amino acid sequence having at least 99.5%, 99%,98.5%, 98%, 98.5% or 97% identity to the amino acid sequence of SEQ IDNO: 55. In one embodiment, RNA encoding a vaccine antigen (i) comprisesthe nucleotide sequence of SEQ ID NO: 56; and/or (ii) encodes an aminoacid sequence comprising the amino acid sequence of SEQ ID NO: 55.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of SEQ ID NO: 57, a nucleotide sequence having atleast 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotidesequence of SEQ ID NO: 57, or a fragment of the nucleotide sequence ofSEQ ID NO: 57, or the nucleotide sequence having at least 99.5%, 99%,98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of SEQ IDNO: 57; and/or (ii) encodes an amino acid sequence comprising the aminoacid sequence of SEQ ID NO: 55, an amino acid sequence having at least99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequenceof SEQ ID NO: 55, or an immunogenic fragment of the amino acid sequenceof SEQ ID NO: 55, or the amino acid sequence having at least 99.5%, 99%,98.5%, 98%, 98.5% or 97% identity to the amino acid sequence of SEQ IDNO: 55. In one embodiment, RNA encoding a vaccine antigen (i) comprisesthe nucleotide sequence of SEQ ID NO: 57; and/or (ii) encodes an aminoacid sequence comprising the amino acid sequence of SEQ ID NO: 55.

In one embodiment, a vaccine antigen comprises the amino acid sequenceof SEQ ID NO: 58, an amino acid sequence having at least 99.5%, 99%,98.5%, 98%, 98.5% or 97% identity to the amino acid sequence of SEQ IDNO: 58, or an immunogenic fragment of the amino acid sequence of SEQ IDNO: 58, or the amino acid sequence having at least 99.5%, 99%, 98.5%,98%, 98.5% or 97% identity to the amino acid sequence of SEQ ID NO: 58.In one embodiment, a vaccine antigen comprises the amino acid sequenceof SEQ ID NO: 58.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of SEQ ID NO: 59, a nucleotide sequence having atleast 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotidesequence of SEQ ID NO: 59, or a fragment of the nucleotide sequence ofSEQ ID NO: 59, or the nucleotide sequence having at least 99.5%, 99%,98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of SEQ IDNO: 59; and/or (ii) encodes an amino acid sequence comprising the aminoacid sequence of SEQ ID NO: 58, an amino acid sequence having at least99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequenceof SEQ ID NO: 58, or an immunogenic fragment of the amino acid sequenceof SEQ ID NO: 58, or the amino acid sequence having at least 99.5%, 99%,98.5%, 98%, 98.5% or 97% identity to the amino acid sequence of SEQ IDNO: 58. In one embodiment, RNA encoding a vaccine antigen (i) comprisesthe nucleotide sequence of SEQ ID NO: 59; and/or (ii) encodes an aminoacid sequence comprising the amino acid sequence of SEQ ID NO: 58.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of SEQ ID NO: 60, a nucleotide sequence having atleast 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotidesequence of SEQ ID NO: 60, or a fragment of the nucleotide sequence ofSEQ ID NO: 60, or the nucleotide sequence having at least 99.5%, 99%,98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of SEQ IDNO: 60; and/or (ii) encodes an amino acid sequence comprising the aminoacid sequence of SEQ ID NO: 58, an amino acid sequence having at least99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequenceof SEQ ID NO: 58, or an immunogenic fragment of the amino acid sequenceof SEQ ID NO: 58, or the amino acid sequence having at least 99.5%, 99%,98.5%, 98%, 98.5% or 97% identity to the amino acid sequence of SEQ IDNO: 58. In one embodiment, RNA encoding a vaccine antigen (i) comprisesthe nucleotide sequence of SEQ ID NO: 60; and/or (ii) encodes an aminoacid sequence comprising the amino acid sequence of SEQ ID NO: 58.

In one embodiment, a vaccine antigen comprises the amino acid sequenceof SEQ ID NO: 61, an amino acid sequence having at least 99.5%, 99%,98.5%, 98%, 98.5% or 97% identity to the amino acid sequence of SEQ IDNO: 61, or an immunogenic fragment of the amino acid sequence of SEQ IDNO: 61, or the amino acid sequence having at least 99.5%, 99%, 98.5%,98%, 98.5% or 97% identity to the amino acid sequence of SEQ ID NO: 61.In one embodiment, a vaccine antigen comprises the amino acid sequenceof SEQ ID NO: 61.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of SEQ ID NO: 62a, a nucleotide sequence having atleast 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotidesequence of SEQ ID NO: 62a, or a fragment of the nucleotide sequence ofSEQ ID NO: 62a, or the nucleotide sequence having at least 99.5%, 99%,98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of SEQ IDNO: 62a; and/or (ii) encodes an amino acid sequence comprising the aminoacid sequence of SEQ ID NO: 61, an amino acid sequence having at least99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequenceof SEQ ID NO: 61, or an immunogenic fragment of the amino acid sequenceof SEQ ID NO: 61, or the amino acid sequence having at least 99.5%, 99%,98.5%, 98%, 98.5% or 97% identity to the amino acid sequence of SEQ IDNO: 61. In one embodiment, RNA encoding a vaccine antigen (i) comprisesthe nucleotide sequence of SEQ ID NO: 62a; and/or (ii) encodes an aminoacid sequence comprising the amino acid sequence of SEQ ID NO: 61.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of SEQ ID NO: 63a, a nucleotide sequence having atleast 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotidesequence of SEQ ID NO: 63a, or a fragment of the nucleotide sequence ofSEQ ID NO: 63a, or the nucleotide sequence having at least 99.5%, 99%,98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of SEQ IDNO: 63a; and/or (ii) encodes an amino acid sequence comprising the aminoacid sequence of SEQ ID NO: 61, an amino acid sequence having at least99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequenceof SEQ ID NO: 61, or an immunogenic fragment of the amino acid sequenceof SEQ ID NO: 61, or the amino acid sequence having at least 99.5%, 99%,98.5%, 98%, 98.5% or 97% identity to the amino acid sequence of SEQ IDNO: 61. In one embodiment, RNA encoding a vaccine antigen (i) comprisesthe nucleotide sequence of SEQ ID NO: 63a; and/or (ii) encodes an aminoacid sequence comprising the amino acid sequence of SEQ ID NO: 61.

In one embodiment, a vaccine antigen comprises the amino acid sequenceof SEQ ID NO: 52, an amino acid sequence having at least 99.5%, 99%,98.5%, 98%, 98.5% or 97% identity to the amino acid sequence of SEQ IDNO: 52, or an immunogenic fragment of the amino acid sequence of SEQ IDNO: 52, or the amino acid sequence having at least 99.5%, 99%, 98.5%,98%, 98.5% or 97% identity to the amino acid sequence of SEQ ID NO: 52.In one embodiment, a vaccine antigen comprises the amino acid sequenceof SEQ ID NO: 52.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of SEQ ID NO: 53, a nucleotide sequence having atleast 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotidesequence of SEQ ID NO: 53, or a fragment of the nucleotide sequence ofSEQ ID NO: 53, or the nucleotide sequence having at least 99.5%, 99%,98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of SEQ IDNO: 53; and/or (ii) encodes an amino acid sequence comprising the aminoacid sequence of SEQ ID NO: 52, an amino acid sequence having at least99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequenceof SEQ ID NO: 52, or an immunogenic fragment of the amino acid sequenceof SEQ ID NO: 52, or the amino acid sequence having at least 99.5%, 99%,98.5%, 98%, 98.5% or 97% identity to the amino acid sequence of SEQ IDNO: 52. In one embodiment, RNA encoding a vaccine antigen (i) comprisesthe nucleotide sequence of SEQ ID NO: 53; and/or (ii) encodes an aminoacid sequence comprising the amino acid sequence of SEQ ID NO: 52.

In one embodiment, RNA encoding a vaccine antigen (i) comprises thenucleotide sequence of SEQ ID NO: 54, a nucleotide sequence having atleast 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotidesequence of SEQ ID NO: 54, or a fragment of the nucleotide sequence ofSEQ ID NO: 54, or the nucleotide sequence having at least 99.5%, 99%,98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of SEQ IDNO: 54; and/or (ii) encodes an amino acid sequence comprising the aminoacid sequence of SEQ ID NO: 52, an amino acid sequence having at least99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequenceof SEQ ID NO: 52, or an immunogenic fragment of the amino acid sequenceof SEQ ID NO: 52, or the amino acid sequence having at least 99.5%, 99%,98.5%, 98%, 98.5% or 97% identity to the amino acid sequence of SEQ IDNO: 52. In one embodiment, RNA encoding a vaccine antigen (i) comprisesthe nucleotide sequence of SEQ ID NO: 54; and/or (ii) encodes an aminoacid sequence comprising the amino acid sequence of SEQ ID NO: 52.

In one embodiment, the vaccine antigens described above comprise acontiguous sequence of SARS-CoV-2 coronavirus spike (S) protein thatconsists of or essentially consists of the above described amino acidsequences derived from SARS-CoV-2 S protein or immunogenic fragmentsthereof (antigenic peptides or proteins) comprised by the vaccineantigens described above. In one embodiment, the vaccine antigensdescribed above comprise a contiguous sequence of SARS-CoV-2 coronavirusspike (S) protein of no more than 220 amino acids, 215 amino acids, 210amino acids, or 205 amino acids.

In one embodiment, RNA encoding a vaccine antigen is nucleoside modifiedmessenger RNA (modRNA) described herein as BNT162b1 (RBP020.3), BNT162b2(RBP020.1 or RBP020.2), or BNT162b3 (e.g., BNT162b3c). In oneembodiment, RNA encoding a vaccine antigen is nucleoside modifiedmessenger RNA (modRNA) described herein as RBP020.2. In one embodiment,RNA encoding a vaccine antigen is nucleoside modified messenger RNA(modRNA) described herein as BNT162b3 (e.g., BNT162b3c).

In one embodiment, RNA encoding a vaccine antigen is nucleoside modifiedmessenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQID NO: 21, a nucleotide sequence having at least 99%, 98%, 97%, 96%,95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:21, and/or (ii) encodes an amino acid sequence comprising the amino acidsequence of SEQ ID NO: 5, or an amino acid sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequenceof SEQ ID NO: 5. In one embodiment, RNA encoding a vaccine antigen isnucleoside modified messenger RNA (modRNA) and (i) comprises thenucleotide sequence of SEQ ID NO: 21; and/or (ii) encodes an amino acidsequence comprising the amino acid sequence of SEQ ID NO: 5.

In one embodiment, RNA encoding a vaccine antigen is nucleoside modifiedmessenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQID NO: 19, or 20, a nucleotide sequence having at least 99%, 98%, 97%,96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ IDNO: 19, or 20, and/or (ii) encodes an amino acid sequence comprising theamino acid sequence of SEQ ID NO: 7, or an amino acid sequence having atleast 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the aminoacid sequence of SEQ ID NO: 7. In one embodiment, RNA encoding a vaccineantigen is nucleoside modified messenger RNA (modRNA) and (i) comprisesthe nucleotide sequence of SEQ ID NO: 19, or 20; and/or (ii) encodes anamino acid sequence comprising the amino acid sequence of SEQ ID NO: 7.

In one embodiment, RNA encoding a vaccine antigen is nucleoside modifiedmessenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQID NO: 20, a nucleotide sequence having at least 99%, 98%, 97%, 96%,95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:20, and/or (ii) encodes an amino acid sequence comprising the amino acidsequence of SEQ ID NO: 7, or an amino acid sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequenceof SEQ ID NO: 7. In one embodiment, RNA encoding a vaccine antigen isnucleoside modified messenger RNA (modRNA) and (i) comprises thenucleotide sequence of SEQ ID NO: 20; and/or (ii) encodes an amino acidsequence comprising the amino acid sequence of SEQ ID NO: 7.

In one embodiment, RNA encoding a vaccine antigen is nucleoside modifiedmessenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQID NO: 30, a nucleotide sequence having at least 99%, 98%, 97%, 96%,95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:30, and/or (ii) encodes an amino acid sequence comprising the amino acidsequence of SEQ ID NO: 29, or an amino acid sequence having at least99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acidsequence of SEQ ID NO: 29. In one embodiment, RNA encoding a vaccineantigen is nucleoside modified messenger RNA (modRNA) and (i) comprisesthe nucleotide sequence of SEQ ID NO: 30; and/or (ii) encodes an aminoacid sequence comprising the amino acid sequence of SEQ ID NO: 29.

In one embodiment, RNA encoding a vaccine antigen is nucleoside modifiedmessenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQID NO: 50, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%,98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 50,and/or (ii) encodes an amino acid sequence comprising the amino acidsequence of SEQ ID NO: 49, or an amino acid sequence having at least99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequenceof SEQ ID NO: 49. In one embodiment, RNA encoding a vaccine antigen isnucleoside modified messenger RNA (modRNA) and (i) comprises thenucleotide sequence of SEQ ID NO: 50; and/or (ii) encodes an amino acidsequence comprising the amino acid sequence of SEQ ID NO: 49.

In one embodiment, RNA encoding a vaccine antigen is nucleoside modifiedmessenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQID NO: 51, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%,98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 51,and/or (ii) encodes an amino acid sequence comprising the amino acidsequence of SEQ ID NO: 49, or an amino acid sequence having at least99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequenceof SEQ ID NO: 49. In one embodiment, RNA encoding a vaccine antigen isnucleoside modified messenger RNA (modRNA) and (i) comprises thenucleotide sequence of SEQ ID NO: 51; and/or (ii) encodes an amino acidsequence comprising the amino acid sequence of SEQ ID NO: 49.

In one embodiment, RNA encoding a vaccine antigen is nucleoside modifiedmessenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQID NO: 57, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%,98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 57,and/or (ii) encodes an amino acid sequence comprising the amino acidsequence of SEQ ID NO: 55, or an amino acid sequence having at least99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequenceof SEQ ID NO: 55. In one embodiment, RNA encoding a vaccine antigen isnucleoside modified messenger RNA (modRNA) and (i) comprises thenucleotide sequence of SEQ ID NO: 57; and/or (ii) encodes an amino acidsequence comprising the amino acid sequence of SEQ ID NO: 55.

In one embodiment, RNA encoding a vaccine antigen is nucleoside modifiedmessenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQID NO: 60, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%,98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 60,and/or (ii) encodes an amino acid sequence comprising the amino acidsequence of SEQ ID NO: 58, or an amino acid sequence having at least99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequenceof SEQ ID NO: 58. In one embodiment, RNA encoding a vaccine antigen isnucleoside modified messenger RNA (modRNA) and (i) comprises thenucleotide sequence of SEQ ID NO: 60; and/or (ii) encodes an amino acidsequence comprising the amino acid sequence of SEQ ID NO: 58.

In one embodiment, RNA encoding a vaccine antigen is nucleoside modifiedmessenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQID NO: 63a, a nucleotide sequence having at least 99.5%, 99%, 98.5%,98%, 98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 63a,and/or (ii) encodes an amino acid sequence comprising the amino acidsequence of SEQ ID NO: 61, or an amino acid sequence having at least99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequenceof SEQ ID NO: 61. In one embodiment, RNA encoding a vaccine antigen isnucleoside modified messenger RNA (modRNA) and (i) comprises thenucleotide sequence of SEQ ID NO: 63a; and/or (ii) encodes an amino acidsequence comprising the amino acid sequence of SEQ ID NO: 61.

In one embodiment, RNA encoding a vaccine antigen is nucleoside modifiedmessenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQID NO: 53, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%,98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 53,and/or (ii) encodes an amino acid sequence comprising the amino acidsequence of SEQ ID NO: 52, or an amino acid sequence having at least99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequenceof SEQ ID NO: 52. In one embodiment, RNA encoding a vaccine antigen isnucleoside modified messenger RNA (modRNA) and (i) comprises thenucleotide sequence of SEQ ID NO: 53; and/or (ii) encodes an amino acidsequence comprising the amino acid sequence of SEQ ID NO: 52.

In one embodiment, RNA encoding a vaccine antigen is nucleoside modifiedmessenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQID NO: 54, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%,98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 54,and/or (ii) encodes an amino acid sequence comprising the amino acidsequence of SEQ ID NO: 52, or an amino acid sequence having at least99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequenceof SEQ ID NO: 52. In one embodiment, RNA encoding a vaccine antigen isnucleoside modified messenger RNA (modRNA) and (i) comprises thenucleotide sequence of SEQ ID NO: 54; and/or (ii) encodes an amino acidsequence comprising the amino acid sequence of SEQ ID NO: 52.

As used herein, the term “vaccine” refers to a composition that inducesan immune response upon inoculation into a subject. In some embodiments,the induced immune response provides protective immunity.

In one embodiment, the RNA encoding the antigen molecule is expressed incells of the subject to provide the antigen molecule. In one embodiment,expression of the antigen molecule is at the cell surface or into theextracellular space. In one embodiment, the antigen molecule ispresented in the context of MHC. In one embodiment, the RNA encoding theantigen molecule is transiently expressed in cells of the subject. Inone embodiment, after administration of the RNA encoding the antigenmolecule, in particular after intramuscular administration of the RNAencoding the antigen molecule, expression of the RNA encoding theantigen molecule in muscle occurs. In one embodiment, afteradministration of the RNA encoding the antigen molecule, expression ofthe RNA encoding the antigen molecule in spleen occurs. In oneembodiment, after administration of the RNA encoding the antigenmolecule, expression of the RNA encoding the antigen molecule in antigenpresenting cells, preferably professional antigen presenting cellsoccurs. In one embodiment, the antigen presenting cells are selectedfrom the group consisting of dendritic cells, macrophages and B cells.In one embodiment, after administration of the RNA encoding the antigenmolecule, no or essentially no expression of the RNA encoding theantigen molecule in lung and/or liver occurs. In one embodiment, afteradministration of the RNA encoding the antigen molecule, expression ofthe RNA encoding the antigen molecule in spleen is at least 5-fold theamount of expression in lung. In some embodiments, the methods andagents, e.g., RNA compositions, described herein followingadministration, in particular following intramuscular administration, toa subject result in delivery of the RNA encoding a vaccine antigen tolymph nodes and/or spleen. In some embodiments, RNA encoding a vaccineantigen is detectable in lymph nodes and/or spleen 6 hours or laterfollowing administration and preferably up to 6 days or longer. In someembodiments, the methods and agents, e.g., mRNA compositions, describedherein following administration, in particular following intramuscularadministration, to a subject result in delivery of the RNA encoding avaccine antigen to B cell follicles, subcapsular sinus, and/or T cellzone, in particular B cell follicles and/or subcapsular sinus of lymphnodes. In some embodiments, the methods and agents, e.g., mRNAcompositions, described herein following administration, in particularfollowing intramuscular administration, to a subject result in deliveryof the RNA encoding a vaccine antigen to B cells (CD19+), subcapsularsinus macrophages (CD169+) and/or dendritic cells (CD11c+) in the T cellzone and intermediary sinus of lymph nodes, in particular to B cells(CD19+) and/or subcapsular sinus macrophages (CD169+) of lymph nodes.

In some embodiments, the methods and agents, e.g., mRNA compositions,described herein following administration, in particular followingintramuscular administration, to a subject result in delivery of the RNAencoding a vaccine antigen to white pulp of spleen.

In some embodiments, the methods and agents, e.g., mRNA compositions,described herein following administration, in particular followingintramuscular administration, to a subject result in delivery of the RNAencoding a vaccine antigen to B cells, DCs (CD11c+), in particular thosesurrounding the B cells, and/or macrophages of spleen, in particular toB cells and/or DCs (CD11c+).

In one embodiment, the vaccine antigen is expressed in lymph node and/orspleen, in particular in the cells of lymph node and/or spleen describedabove.

The peptide and protein antigens suitable for use according to thepresent disclosure typically include a peptide or protein comprising anepitope of SARS-CoV-2 S protein or a functional variant thereof forinducing an immune response. The peptide or protein or epitope may bederived from a target antigen, i.e. the antigen against which an immuneresponse is to be elicited. For example, the peptide or protein antigenor the epitope contained within the peptide or protein antigen may be atarget antigen or a fragment or variant of a target antigen. The targetantigen may be a coronavirus S protein, in particular SARS-CoV-2 Sprotein.

The antigen molecule or a procession product thereof, e.g., a fragmentthereof, may bind to an antigen receptor such as a BCR or TCR carried byimmune effector cells, or to antibodies. A peptide and protein antigenwhich is provided to a subject according to the present disclosure byadministering RNA encoding the peptide and protein antigen, i.e., avaccine antigen, preferably results in the induction of an immuneresponse, e.g., a humoral and/or cellular immune response in the subjectbeing provided the peptide or protein antigen. Said immune response ispreferably directed against a target antigen, in particular coronavirusS protein, in particular SARS-CoV-2 S protein. Thus, a vaccine antigenmay comprise the target antigen, a variant thereof, or a fragmentthereof. In one embodiment, such fragment or variant is immunologicallyequivalent to the target antigen. In the context of the presentdisclosure, the term “fragment of an antigen” or “variant of an antigen”means an agent which results in the induction of an immune responsewhich immune response targets the antigen, i.e. a target antigen. Thus,the vaccine antigen may correspond to or may comprise the targetantigen, may correspond to or may comprise a fragment of the targetantigen or may correspond to or may comprise an antigen which ishomologous to the target antigen or a fragment thereof. Thus, accordingto the present disclosure, a vaccine antigen may comprise an immunogenicfragment of a target antigen or an amino acid sequence being homologousto an immunogenic fragment of a target antigen. An “immunogenic fragmentof an antigen” according to the present disclosure preferably relates toa fragment of an antigen which is capable of inducing an immune responseagainst the target antigen. The vaccine antigen may be a recombinantantigen.

The term “immunologically equivalent” means that the immunologicallyequivalent molecule such as the immunologically equivalent amino acidsequence exhibits the same or essentially the same immunologicalproperties and/or exerts the same or essentially the same immunologicaleffects, e.g., with respect to the type of the immunological effect. Inthe context of the present disclosure, the term “immunologicallyequivalent” is preferably used with respect to the immunological effectsor properties of antigens or antigen variants used for immunization. Forexample, an amino acid sequence is immunologically equivalent to areference amino acid sequence if said amino acid sequence when exposedto the immune system of a subject induces an immune reaction having aspecificity of reacting with the reference amino acid sequence.

“Activation” or “stimulation”, as used herein, refers to the state of animmune effector cell such as T cell that has been sufficientlystimulated to induce detectable cellular proliferation. Activation canalso be associated with initiation of signaling pathways, inducedcytokine production, and detectable effector functions. The term“activated immune effector cells” refers to, among other things, immuneeffector cells that are undergoing cell division.

The term “priming” refers to a process wherein an immune effector cellsuch as a T cell has its first contact with its specific antigen andcauses differentiation into effector cells such as effector T cells.

The term “clonal expansion” or “expansion” refers to a process wherein aspecific entity is multiplied. In the context of the present disclosure,the term is preferably used in the context of an immunological responsein which immune effector cells are stimulated by an antigen,proliferate, and the specific immune effector cell recognizing saidantigen is amplified. Preferably, clonal expansion leads todifferentiation of the immune effector cells.

The term “antigen” relates to an agent comprising an epitope againstwhich an immune response can be generated. The term “antigen” includes,in particular, proteins and peptides. In one embodiment, an antigen ispresented by cells of the immune system such as antigen presenting cellslike dendritic cells or macrophages. An antigen or a procession productthereof such as a T-cell epitope is in one embodiment bound by a T- orB-cell receptor, or by an immunoglobulin molecule such as an antibody.Accordingly, an antigen or a procession product thereof may reactspecifically with antibodies or T lymphocytes (T cells). In oneembodiment, an antigen is a viral antigen, such as a coronavirus Sprotein, e.g., SARS-CoV-2 S protein, and an epitope is derived from suchantigen.

The term “viral antigen” refers to any viral component having antigenicproperties, i.e. being able to provoke an immune response in anindividual. The viral antigen may be coronavirus S protein, e.g.,SARS-CoV-2 S protein.

The term “expressed on the cell surface” or “associated with the cellsurface” means that a molecule such as an antigen is associated with andlocated at the plasma membrane of a cell, wherein at least a part of themolecule faces the extracellular space of said cell and is accessiblefrom the outside of said cell, e.g., by antibodies located outside thecell. In this context, a part is preferably at least 4, preferably atleast 8, preferably at least 12, more preferably at least 20 aminoacids. The association may be direct or indirect. For example, theassociation may be by one or more transmembrane domains, one or morelipid anchors, or by the interaction with any other protein, lipid,saccharide, or other structure that can be found on the outer leaflet ofthe plasma membrane of a cell. For example, a molecule associated withthe surface of a cell may be a transmembrane protein having anextracellular portion or may be a protein associated with the surface ofa cell by interacting with another protein that is a transmembraneprotein.

“Cell surface” or “surface of a cell” is used in accordance with itsnormal meaning in the art, and thus includes the outside of the cellwhich is accessible to binding by proteins and other molecules. Anantigen is expressed on the surface of cells if it is located at thesurface of said cells and is accessible to binding by e.g.antigen-specific antibodies added to the cells.

The term “extracellular portion” or “exodomain” in the context of thepresent disclosure refers to a part of a molecule such as a protein thatis facing the extracellular space of a cell and preferably is accessiblefrom the outside of said cell, e.g., by binding molecules such asantibodies located outside the cell. Preferably, the term refers to oneor more extracellular loops or domains or a fragment thereof.

The term “epitope” refers to a part or fragment of a molecule such as anantigen that is recognized by the immune system. For example, theepitope may be recognized by T cells, B cells or antibodies. An epitopeof an antigen may include a continuous or discontinuous portion of theantigen and may be between about 5 and about 100, such as between about5 and about 50, more preferably between about 8 and about 30, mostpreferably between about 8 and about 25 amino acids in length, forexample, the epitope may be preferably 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In oneembodiment, an epitope is between about 10 and about 25 amino acids inlength. The term “epitope” includes T cell epitopes.

The term “T cell epitope” refers to a part or fragment of a protein thatis recognized by a T cell when presented in the context of MHCmolecules. The term “major histocompatibility complex” and theabbreviation “MHC” includes MHC class I and MHC class II molecules andrelates to a complex of genes which is present in all vertebrates. MHCproteins or molecules are important for signaling between lymphocytesand antigen presenting cells or diseased cells in immune reactions,wherein the MHC proteins or molecules bind peptide epitopes and presentthem for recognition by T cell receptors on T cells. The proteinsencoded by the MHC are expressed on the surface of cells, and displayboth self-antigens (peptide fragments from the cell itself) andnon-self-antigens (e.g., fragments of invading microorganisms) to a Tcell. In the case of class I MHC/peptide complexes, the binding peptidesare typically about 8 to about 10 amino acids long although longer orshorter peptides may be effective. In the case of class II MHC/peptidecomplexes, the binding peptides are typically about 10 to about 25 aminoacids long and are in particular about 13 to about 18 amino acids long,whereas longer and shorter peptides may be effective.

The peptide and protein antigen can be 2-100 amino acids, including forexample, 5 amino acids, 10 amino acids, 15 amino acids, 20 amino acids,25 amino acids, 30 amino acids, 35 amino acids, 40 amino acids, 45 aminoacids, or 50 amino acids in length. In some embodiments, a peptide canbe greater than 50 amino acids. In some embodiments, the peptide can begreater than 100 amino acids.

The peptide or protein antigen can be any peptide or protein that caninduce or increase the ability of the immune system to developantibodies and T cell responses to the peptide or protein.

In one embodiment, vaccine antigen is recognized by an immune effectorcell. Preferably, the vaccine antigen if recognized by an immuneeffector cell is able to induce in the presence of appropriateco-stimulatory signals, stimulation, priming and/or expansion of theimmune effector cell carrying an antigen receptor recognizing thevaccine antigen. In the context of the embodiments of the presentdisclosure, the vaccine antigen is preferably presented or present onthe surface of a cell, preferably an antigen presenting cell. In oneembodiment, an antigen is presented by a diseased cell such as avirus-infected cell. In one embodiment, an antigen receptor is a TCRwhich binds to an epitope of an antigen presented in the context of MHC.In one embodiment, binding of a TCR when expressed by T cells and/orpresent on T cells to an antigen presented by cells such as antigenpresenting cells results in stimulation, priming and/or expansion ofsaid T cells. In one embodiment, binding of a TCR when expressed by Tcells and/or present on T cells to an antigen presented on diseasedcells results in cytolysis and/or apoptosis of the diseased cells,wherein said T cells preferably release cytotoxic factors, e.g.perforins and granzymes.

In one embodiment, an antigen receptor is an antibody or B cell receptorwhich binds to an epitope in an antigen. In one embodiment, an antibodyor B cell receptor binds to native epitopes of an antigen.

Nucleic Acids

The term “polynucleotide” or “nucleic acid”, as used herein, is intendedto include DNA and RNA such as genomic DNA, cDNA, mRNA, recombinantlyproduced and chemically synthesized molecules. A nucleic acid may besingle-stranded or double-stranded. RNA includes in vitro transcribedRNA (IVT RNA) or synthetic RNA. According to the present disclosure, apolynucleotide is preferably isolated.

Nucleic acids may be comprised in a vector. The term “vector” as usedherein includes any vectors known to the skilled person includingplasmid vectors, cosmid vectors, phage vectors such as lambda phage,viral vectors such as retroviral, adenoviral or baculoviral vectors, orartificial chromosome vectors such as bacterial artificial chromosomes(BAC), yeast artificial chromosomes (YAC), or P1 artificial chromosomes(PAC). Said vectors include expression as well as cloning vectors.Expression vectors comprise plasmids as well as viral vectors andgenerally contain a desired coding sequence and appropriate DNAsequences necessary for the expression of the operably linked codingsequence in a particular host organism (e.g., bacteria, yeast, plant,insect, or mammal) or in in vitro expression systems. Cloning vectorsare generally used to engineer and amplify a certain desired DNAfragment and may lack functional sequences needed for expression of thedesired DNA fragments.

In one embodiment of all aspects of the present disclosure, the RNAencoding the vaccine antigen is expressed in cells such as antigenpresenting cells of the subject treated to provide the vaccine antigen.

The nucleic acids described herein may be recombinant and/or isolatedmolecules.

In the present disclosure, the term “RNA” relates to a nucleic acidmolecule which includes ribonucleotide residues. In preferredembodiments, the RNA contains all or a majority of ribonucleotideresidues. As used herein, “ribonucleotide” refers to a nucleotide with ahydroxyl group at the 2′-position of a β-D-ribofuranosyl group. RNAencompasses without limitation, double stranded RNA, single strandedRNA, isolated RNA such as partially purified RNA, essentially pure RNA,synthetic RNA, recombinantly produced RNA, as well as modified RNA thatdiffers from naturally occurring RNA by the addition, deletion,substitution and/or alteration of one or more nucleotides. Suchalterations may refer to addition of non-nucleotide material to internalRNA nucleotides or to the end(s) of RNA. It is also contemplated hereinthat nucleotides in RNA may be non-standard nucleotides, such aschemically synthesized nucleotides or deoxynucleotides. For the presentdisclosure, these altered RNAs are considered analogs ofnaturally-occurring RNA.

In certain embodiments of the present disclosure, the RNA is messengerRNA (mRNA) that relates to a RNA transcript which encodes a peptide orprotein. As established in the art, mRNA generally contains a 5′untranslated region (5′-UTR), a peptide coding region and a 3′untranslated region (3′-UTR). In some embodiments, the RNA is producedby in vitro transcription or chemical synthesis. In one embodiment, themRNA is produced by in vitro transcription using a DNA template whereDNA refers to a nucleic acid that contains deoxyribonucleotides.

In one embodiment, RNA is in vitro transcribed RNA (IVT-RNA) and may beobtained by in vitro transcription of an appropriate DNA template. Thepromoter for controlling transcription can be any promoter for any RNApolymerase. A DNA template for in vitro transcription may be obtained bycloning of a nucleic acid, in particular cDNA, and introducing it intoan appropriate vector for in vitro transcription. The cDNA may beobtained by reverse transcription of RNA. In certain embodiments of thepresent disclosure, the RNA is “replicon RNA” or simply a “replicon”, inparticular “self-replicating RNA” or “self-amplifying RNA”. In oneparticularly preferred embodiment, the replicon or self-replicating RNAis derived from or comprises elements derived from a ssRNA virus, inparticular a positive-stranded ssRNA virus such as an alphavirus.Alphaviruses are typical representatives of positive-stranded RNAviruses. Alphaviruses replicate in the cytoplasm of infected cells (forreview of the alphaviral life cycle see José et al., Future Microbiol.,2009, vol. 4, pp. 837-856). The total genome length of many alphavirusestypically ranges between 11,000 and 12,000 nucleotides, and the genomicRNA typically has a 5′-cap, and a 3′ poly(A) tail. The genome ofalphaviruses encodes non-structural proteins (involved in transcription,modification and replication of viral RNA and in protein modification)and structural proteins (forming the virus particle). There aretypically two open reading frames (ORFs) in the genome. The fournon-structural proteins (nsP1-nsP4) are typically encoded together by afirst ORF beginning near the 5′ terminus of the genome, while alphavirusstructural proteins are encoded together by a second ORF which is founddownstream of the first ORF and extends near the 3′ terminus of thegenome. Typically, the first ORF is larger than the second ORF, theratio being roughly 2:1. In cells infected by an alphavirus, only thenucleic acid sequence encoding non-structural proteins is translatedfrom the genomic RNA, while the genetic information encoding structuralproteins is translatable from a subgenomic transcript, which is an RNAmolecule that resembles eukaryotic messenger RNA (mRNA; Gould et al.,2010, Antiviral Res., vol. 87 pp. 111-124). Following infection, i.e. atearly stages of the viral life cycle, the (+) stranded genomic RNAdirectly acts like a messenger RNA for the translation of the openreading frame encoding the non-structural poly-protein (nsP1234).Alphavirus-derived vectors have been proposed for delivery of foreigngenetic information into target cells or target organisms. In simpleapproaches, the open reading frame encoding alphaviral structuralproteins is replaced by an open reading frame encoding a protein ofinterest. Alphavirus-based trans-replication systems rely on alphavirusnucleotide sequence elements on two separate nucleic acid molecules: onenucleic acid molecule encodes a viral replicase, and the other nucleicacid molecule is capable of being replicated by said replicase in trans(hence the designation trans-replication system). Trans-replicationrequires the presence of both these nucleic acid molecules in a givenhost cell. The nucleic acid molecule capable of being replicated by thereplicase in trans must comprise certain alphaviral sequence elements toallow recognition and RNA synthesis by the alphaviral replicase.

In one embodiment, the RNA described herein may have modifiednucleosides. In some embodiments, the RNA comprises a modifiednucleoside in place of at least one (e.g., every) uridine.

The term “uracil,” as used herein, describes one of the nucleobases thatcan occur in the nucleic acid of RNA. The structure of uracil is:

The term “uridine,” as used herein, describes one of the nucleosidesthat can occur in RNA.

The structure of uridine is:

UTP (uridine 5′-triphosphate) has the following structure:

Pseudo-UTP (pseudouridine 5′-triphosphate) has the following structure:

“Pseudouridine” is one example of a modified nucleoside that is anisomer of uridine, where the uracil is attached to the pentose ring viaa carbon-carbon bond instead of a nitrogen-carbon glycosidic bond.

Another exemplary modified nucleoside is N1-methyl-pseudouridine (m1ψ),which has the structure:

N1-methyl-pseudo-UTP has the following structure:

Another exemplary modified nucleoside is 5-methyl-uridine (m5U), whichhas the structure:

In some embodiments, one or more uridine in the RNA described herein isreplaced by a modified nucleoside. In some embodiments, the modifiednucleoside is a modified uridine.

In some embodiments, RNA comprises a modified nucleoside in place of atleast one uridine.

In some embodiments, RNA comprises a modified nucleoside in place ofeach uridine.

In some embodiments, the modified nucleoside is independently selectedfrom pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and5-methyl-uridine (m5U). In some embodiments, the modified nucleosidecomprises pseudouridine (ψ). In some embodiments, the modifiednucleoside comprises N1-methyl-pseudouridine (m1ψ). In some embodiments,the modified nucleoside comprises 5-methyl-uridine (m5U). In someembodiments, RNA may comprise more than one type of modified nucleoside,and the modified nucleosides are independently selected frompseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine(m5U). In some embodiments, the modified nucleosides comprisepseudouridine (ψ) and N1-methyl-pseudouridine (m1ψ). In someembodiments, the modified nucleosides comprise pseudouridine (ψ) and5-methyl-uridine (m5U). In some embodiments, the modified nucleosidescomprise N1-methyl-pseudouridine (m1ψ) and 5-methyl-uridine (m5U). Insome embodiments, the modified nucleosides comprise pseudouridine (ψ),N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U).

In some embodiments, the modified nucleoside replacing one or more,e.g., all, uridine in the RNA may be any one or more of 3-methyl-uridine(m³U), 5-methoxy-uridine (mo⁵U), 5-aza-uridine, 6-aza-uridine,2-thio-5-aza-uridine, 2-thio-uridine (s²U), 4-thio-uridine (s⁴U),4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho⁵U),5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or5-bromo-uridine), uridine 5-oxyacetic acid (cmo⁵U), uridine 5-oxyaceticacid methyl ester (mcmo⁵U), 5-carboxymethyl-uridine (cm⁵U),1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm⁵U),5-carboxyhydroxymethyl-uridine methyl ester (mchm⁵U),5-methoxycarbonylmethyl-uridine (mcm⁵U),5-methoxycarbonylmethyl-2-thio-uridine (mcm⁵s²U),5-aminomethyl-2-thio-uridine (nm⁵s²U), 5-methylaminomethyl-uridine(mnm⁵U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine(mnm⁵s²U), 5-methylaminomethyl-2-seleno-uridine (mnm⁵se2U),5-carbamoylmethyl-uridine (ncm⁵U), 5-carboxymethylaminomethyl-uridine(cmnm⁵U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm⁵s²U),5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine(τm⁵U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine(τm5s2U), 1-taurinomethyl-4-thio-pseudouridine), 5-methyl-2-thio-uridine(m⁵s²U), 1-methyl-4-thio-pseudouridine (m¹s⁴ψ),4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m³ψ),2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D),dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m⁵D),2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine,2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine,4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine,3-(3-amino-3-carboxypropyl)uridine (acp³U),1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp³ ψ),5-(isopentenylaminomethyl)uridine (inm⁵U),5-(isopentenylaminomethyl)-2-thio-uridine (inm⁵s²U), α-thio-uridine,2′-O-methyl-uridine (Um), 5,2′-O-dimethyl-uridine (m⁵Um),2′-O-methyl-pseudouridine (4m), 2-thio-2′-O-methyl-uridine (s²Um),5-methoxycarbonylmethyl-2′-O-methyl-uridine (mcm⁵Um),5-carbamoylmethyl-2′-O-methyl-uridine (ncm⁵Um),5-carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm⁵Um),3,2′-O-dimethyl-uridine (m³Um),5-(isopentenylaminomethyl)-2′-O-methyl-uridine (inm⁵Um), 1-thio-uridine,deoxythymidine, 2′-F-ara-uridine, 2′-F-uridine, 2′-OH-ara-uridine,5-(2-carbomethoxyvinyl) uridine, 5-[3-(1-E-propenylamino)uridine, or anyother modified uridine known in the art.

In one embodiment, the RNA comprises other modified nucleosides orcomprises further modified nucleosides, e.g., modified cytidine. Forexample, in one embodiment, in the RNA 5-methylcytidine is substitutedpartially or completely, preferably completely, for cytidine. In oneembodiment, the RNA comprises 5-methylcytidine and one or more selectedfrom pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and5-methyl-uridine (m5U). In one embodiment, the RNA comprises5-methylcytidine and N1-methyl-pseudouridine (m1ψ). In some embodiments,the RNA comprises 5-methylcytidine in place of each cytidine andN1-methyl-pseudouridine (m1ψ)) in place of each uridine.

In some embodiments, the RNA according to the present disclosurecomprises a 5′-cap. In one embodiment, the RNA of the present disclosuredoes not have uncapped 5′-triphosphates. In one embodiment, the RNA maybe modified by a 5′-cap analog. The term “5′-cap” refers to a structurefound on the 5′-end of an mRNA molecule and generally consists of aguanosine nucleotide connected to the mRNA via a 5′- to 5′-triphosphatelinkage. In one embodiment, this guanosine is methylated at the7-position. Providing an RNA with a 5′-cap or 5′-cap analog may beachieved by in vitro transcription, in which the 5′-cap isco-transcriptionally expressed into the RNA strand, or may be attachedto RNA post-transcriptionally using capping enzymes. In someembodiments, the RNA (e.g., mRNA) comprises a cap0, cap1, or cap2,preferably cap1 or cap2, more preferably cap1. According to the presentdisclosure, the term “cap0” comprises the structure “m⁷GpppN”, wherein Nis any nucleoside bearing an OH moiety at position 2′. According to thepresent disclosure, the term “cap1” comprises the structure “m⁷GpppNm”,wherein Nm is any nucleoside bearing an OCH₃ moiety at position 2′.According to the present disclosure, the term “cap2” comprises thestructure “m⁷GpppNmNm”, wherein each Nm is independently any nucleosidebearing an OCH₃ moiety at position 2′.

In some embodiments, the building block cap for RNA is m₂^(7,3′-O)-Gppp(m₁ ^(2′-O))ApG (also sometimes referred to as m₂^(7,3′O)G(5′)ppp(5′)m^(2′-O)ApG), which has the following structure:

Below is an exemplary Cap1 RNA, which comprises RNA and m₂^(7,3′O)G(5′)ppp(5′)m^(2′-O)ApG:

Below is another exemplary Cap1 RNA (no cap analog):

In some embodiments, the RNA is modified with “Cap0” structures using,in one embodiment, the cap analog anti-reverse cap (ARCA Cap (m₂^(7,3′O)G(5′)G)) with the structure:

Below is an exemplary Cap0 RNA comprising RNA and m₂^(7,3′O)G(5′)ppp(5′)G:

In some embodiments, the “Cap0” structures are generated using the capanalog Beta-S-ARCA (m₂ ^(7,2′O)G(5′)ppSp(5′)G) with the structure:

Below is an exemplary Cap0 RNA comprising Beta-S-ARCA (m₂^(7,2′O)G(5′)ppSp(5′)G) and RNA:

The “D1” diastereomer of beta-S-ARCA or “beta-S-ARCA(D1)” is thediastereomer of beta-S-ARCA which elutes first on an HPLC columncompared to the D2 diastereomer of beta-S-ARCA (beta-S-ARCA(D2)) andthus exhibits a shorter retention time (cf., WO 2011/015347, hereinincorporated by reference).

A particularly preferred cap is beta-S-ARCA(D1) (m₂ ^(7,2′-O)GppSpG) orm₂ ^(7,3′-O)Gppp(m₁ ^(2′-O))ApG. In some embodiments, RNA according tothe present disclosure comprises a 5′-UTR and/or a 3′-UTR. The term“untranslated region” or “UTR” relates to a region in a DNA moleculewhich is transcribed but is not translated into an amino acid sequence,or to the corresponding region in an RNA molecule, such as an mRNAmolecule. An untranslated region (UTR) can be present 5′ (upstream) ofan open reading frame (5′-UTR) and/or 3′ (downstream) of an open readingframe (3′-UTR). A 5′-UTR, if present, is located at the 5′ end, upstreamof the start codon of a protein-encoding region. A 5′-UTR is downstreamof the 5′-cap (if present), e.g. directly adjacent to the 5′-cap. A3′-UTR, if present, is located at the 3′ end, downstream of thetermination codon of a protein-encoding region, but the term “3′-UTR”does preferably not include the poly(A) sequence. Thus, the 3′-UTR isupstream of the poly(A) sequence (if present), e.g. directly adjacent tothe poly(A) sequence.

In some embodiments, RNA comprises a 5′-UTR comprising the nucleotidesequence of SEQ ID NO: 12, or a nucleotide sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequenceof SEQ ID NO: 12.

In some embodiments, RNA comprises a 3′-UTR comprising the nucleotidesequence of SEQ ID NO: 13, or a nucleotide sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequenceof SEQ ID NO: 13.

A particularly preferred 5′-UTR comprises the nucleotide sequence of SEQID NO: 12. A particularly preferred 3′-UTR comprises the nucleotidesequence of SEQ ID NO: 13.

In some embodiments, the RNA according to the present disclosurecomprises a 3′-poly(A) sequence.

As used herein, the term “poly(A) sequence” or “poly-A tail” refers toan uninterrupted or interrupted sequence of adenylate residues which istypically located at the 3′-end of an RNA molecule. Poly(A) sequencesare known to those of skill in the art and may follow the 3′-UTR in theRNAs described herein. An uninterrupted poly(A) sequence ischaracterized by consecutive adenylate residues. In nature, anuninterrupted poly(A) sequence is typical. RNAs disclosed herein canhave a poly(A) sequence attached to the free 3′-end of the RNA by atemplate-independent RNA polymerase after transcription or a poly(A)sequence encoded by DNA and transcribed by a template-dependent RNApolymerase.

It has been demonstrated that a poly(A) sequence of about 120 Anucleotides has a beneficial influence on the levels of RNA intransfected eukaryotic cells, as well as on the levels of protein thatis translated from an open reading frame that is present upstream (5′)of the poly(A) sequence (Holtkamp et al., 2006, Blood, vol. 108, pp.4009-4017).

The poly(A) sequence may be of any length. In some embodiments, apoly(A) sequence comprises, essentially consists of, or consists of atleast 20, at least 30, at least 40, at least 80, or at least 100 and upto 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides,and, in particular, about 120 A nucleotides. In this context,“essentially consists of” means that most nucleotides in the poly(A)sequence, typically at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% by number of nucleotides in the poly(A) sequence are A nucleotides,but permits that remaining nucleotides are nucleotides other than Anucleotides, such as U nucleotides (uridylate), G nucleotides(guanylate), or C nucleotides (cytidylate). In this context, “consistsof” means that all nucleotides in the poly(A) sequence, i.e., 100% bynumber of nucleotides in the poly(A) sequence, are A nucleotides. Theterm “A nucleotide” or “A” refers to adenylate.

In some embodiments, a poly(A) sequence is attached during RNAtranscription, e.g., during preparation of in vitro transcribed RNA,based on a DNA template comprising repeated dT nucleotides(deoxythymidylate) in the strand complementary to the coding strand. TheDNA sequence encoding a poly(A) sequence (coding strand) is referred toas poly(A) cassette.

In some embodiments, the poly(A) cassette present in the coding strandof DNA essentially consists of dA nucleotides, but is interrupted by arandom sequence of the four nucleotides (dA, dC, dG, and dT). Suchrandom sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides inlength. Such a cassette is disclosed in WO 2016/005324 A1, herebyincorporated by reference. Any poly(A) cassette disclosed in WO2016/005324 A1 may be used in the present disclosure. A poly(A) cassettethat essentially consists of dA nucleotides, but is interrupted by arandom sequence having an equal distribution of the four nucleotides(dA, dC, dG, dT) and having a length of e.g., 5 to 50 nucleotides shows,on DNA level, constant propagation of plasmid DNA in E. coli and isstill associated, on RNA level, with the beneficial properties withrespect to supporting RNA stability and translational efficiency isencompassed. Consequently, in some embodiments, the poly(A) sequencecontained in an RNA molecule described herein essentially consists of Anucleotides, but is interrupted by a random sequence of the fournucleotides (A, C, G, U). Such random sequence may be 5 to 50, 10 to 30,or 10 to 20 nucleotides in length.

In some embodiments, no nucleotides other than A nucleotides flank apoly(A) sequence at its 3′-end, i.e., the poly(A) sequence is not maskedor followed at its 3′-end by a nucleotide other than A.

In some embodiments, the poly(A) sequence may comprise at least 20, atleast 30, at least 40, at least 80, or at least 100 and up to 500, up to400, up to 300, up to 200, or up to 150 nucleotides. In someembodiments, the poly(A) sequence may essentially consist of at least20, at least 30, at least 40, at least 80, or at least 100 and up to500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In someembodiments, the poly(A) sequence may consist of at least 20, at least30, at least 40, at least 80, or at least 100 and up to 500, up to 400,up to 300, up to 200, or up to 150 nucleotides. In some embodiments, thepoly(A) sequence comprises at least 100 nucleotides. In someembodiments, the poly(A) sequence comprises about 150 nucleotides. Insome embodiments, the poly(A) sequence comprises about 120 nucleotides.

In some embodiments, a poly(A) sequence included in an RNA describedherein is a interrupted poly(A) sequence, e.g., as described inWO2016/005324, the entire content of which is incorporated herein byreference for purposes described herein. In some embodiments, a poly(A)sequence comprises a stretch of at least 20 adenosine residues(including, e.g., at least 30, at least 40, at least 50, at least 60, atleast 70, or more adenosine residues), followed by a linker sequence(e.g., in some embodiments comprising non-A nucleotides) and anotherstretch of at least 20 adenosine residues (including, e.g., at least 30,at least 40, at least 50, at least 60, at least 70, or more adenosineresidues). In some embodiments, such a linker sequence may be 3-50nucleotides in length, or 5-25 nucleotides in length, or 10-15nucleotides in length. In some embodiments, a poly(A) sequence comprisesa stretch of about 30 adenosine residues, followed by a linker sequencehaving a length of about 5-15 nucleotides (e.g., in some embodimentscomprising non-A nucleotides) and another stretch of about 70 adenosineresidues.

In some embodiments, RNA comprises a poly(A) sequence comprising thenucleotide sequence of SEQ ID NO: 14, or a nucleotide sequence having atleast 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to thenucleotide sequence of SEQ ID NO: 14.

A particularly preferred poly(A) sequence comprises the nucleotidesequence of SEQ ID NO: 14.

According to the present disclosure, vaccine antigen is preferablyadministered as single-stranded, 5′-capped RNA (e.g., mRNA) that istranslated into the respective protein upon entering cells of a subjectbeing administered the RNA. Preferably, the RNA contains structuralelements optimized for maximal efficacy of the RNA with respect tostability and translational efficiency (5′-cap, 5′-UTR, 3′-UTR, poly(A)sequence).

In one embodiment, beta-S-ARCA(D1) is utilized as specific cappingstructure at the 5′-end of the RNA. In one embodiment, m₂^(7,3′-O)Gppp(m₁ ^(2′-O))ApG is utilized as specific capping structureat the 5′-end of the RNA. In one embodiment, the 5′-UTR sequence isderived from the human alpha-globin mRNA and optionally has an optimized‘Kozak sequence’ to increase translational efficiency. In oneembodiment, a combination of two sequence elements (FI element) derivedfrom the “amino terminal enhancer of split” (AES) mRNA (called F) andthe mitochondrial encoded 125 ribosomal RNA (called 1) are placedbetween the coding sequence and the poly(A) sequence to assure highermaximum protein levels and prolonged persistence of the RNA (e.g.,mRNA). In one embodiment, two re-iterated 3′-UTRs derived from the humanbeta-globin mRNA are placed between the coding sequence and the poly(A)sequence to assure higher maximum protein levels and prolongedpersistence of the RNA (e.g., mRNA). In one embodiment, a poly(A)sequence measuring 110 nucleotides in length, consisting of a stretch of30 adenosine residues, followed by a linker sequence (e.g., 10nucleotide linker sequence) and another 70 adenosine residues is used.This poly(A) sequence was designed to enhance RNA stability andtranslational efficiency.

In one embodiment of all aspects of the present disclosure, RNA encodinga vaccine antigen is expressed in cells of the subject treated toprovide the vaccine antigen. In one embodiment of all aspects of thepresent disclosure, the RNA is transiently expressed in cells of thesubject. In one embodiment of all aspects of the present disclosure, theRNA is in vitro transcribed RNA. In one embodiment of all aspects of thepresent disclosure, expression of the vaccine antigen is at the cellsurface. In one embodiment of all aspects of the present disclosure, thevaccine antigen is expressed and presented in the context of MHC. In oneembodiment of all aspects of the present disclosure, expression of thevaccine antigen is into the extracellular space, i.e., the vaccineantigen is secreted.

In the context of the present disclosure, the term “transcription”relates to a process, wherein the genetic code in a DNA sequence istranscribed into RNA. Subsequently, the RNA may be translated intopeptide or protein.

According to the present disclosure, the term “transcription” comprises“in vitro transcription”, wherein the term “in vitro transcription”relates to a process wherein RNA, in particular mRNA, is in vitrosynthesized in a cell-free system, preferably using appropriate cellextracts. Preferably, cloning vectors are applied for the generation oftranscripts. These cloning vectors are generally designated astranscription vectors and are according to the present disclosureencompassed by the term “vector”. According to the present disclosure,the RNA used in the present disclosure preferably is in vitrotranscribed RNA (IVT-RNA) and may be obtained by in vitro transcriptionof an appropriate DNA template. The promoter for controllingtranscription can be any promoter for any RNA polymerase. Particularexamples of RNA polymerases are the T7, T3, and SP6 RNA polymerases.Preferably, the in vitro transcription according to the disclosure iscontrolled by a T7 or SP6 promoter. A DNA template for in vitrotranscription may be obtained by cloning of a nucleic acid, inparticular cDNA, and introducing it into an appropriate vector for invitro transcription. The cDNA may be obtained by reverse transcriptionof RNA.

With respect to RNA, the term “expression” or “translation” relates tothe process in the ribosomes of a cell by which a strand of mRNA directsthe assembly of a sequence of amino acids to make a peptide or protein.

In one embodiment, after administration of the RNA described herein,e.g., formulated as RNA lipid particles, at least a portion of the RNAis delivered to a target cell. In one embodiment, at least a portion ofthe RNA is delivered to the cytosol of the target cell. In oneembodiment, the RNA is translated by the target cell to produce thepeptide or protein it encodes. In one embodiment, the target cell is aspleen cell. In one embodiment, the target cell is an antigen presentingcell such as a professional antigen presenting cell in the spleen. Inone embodiment, the target cell is a dendritic cell or macrophage. RNAparticles such as RNA lipid particles described herein may be used fordelivering RNA to such target cell. Accordingly, the present disclosurealso relates to a method for delivering RNA to a target cell in asubject comprising the administration of the RNA particles describedherein to the subject. In one embodiment, the RNA is delivered to thecytosol of the target cell. In one embodiment, the RNA is translated bythe target cell to produce the peptide or protein encoded by the RNA.“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

In one embodiment, the RNA encoding vaccine antigen to be administeredaccording to the present disclosure is non-immunogenic. RNA encodingimmunostimulant may be administered according to the present disclosureto provide an adjuvant effect. The RNA encoding immunostimulant may bestandard RNA or non-immunogenic RNA.

The term “non-immunogenic RNA” as used herein refers to RNA that doesnot induce a response by the immune system upon administration, e.g., toa mammal, or induces a weaker response than would have been induced bythe same RNA that differs only in that it has not been subjected to themodifications and treatments that render the non-immunogenic RNAnon-immunogenic, i.e., than would have been induced by standard RNA(stdRNA). In one preferred embodiment, non-immunogenic RNA, which isalso termed modified RNA (modRNA) herein, is rendered non-immunogenic byincorporating modified nucleosides suppressing RNA-mediated activationof innate immune receptors into the RNA and removing double-stranded RNA(dsRNA).

For rendering the non-immunogenic RNA non-immunogenic by theincorporation of modified nucleosides, any modified nucleoside may beused as long as it lowers or suppresses immunogenicity of the RNA.Particularly preferred are modified nucleosides that suppressRNA-mediated activation of innate immune receptors. In one embodiment,the modified nucleosides comprises a replacement of one or more uridineswith a nucleoside comprising a modified nucleobase. In one embodiment,the modified nucleobase is a modified uracil. In one embodiment, thenucleoside comprising a modified nucleobase is selected from the groupconsisting of 3-methyl-uridine (m³U), 5-methoxy-uridine (mo¹U),5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine(s²U), 4-thio-uridine (s⁴U), 4-thio-pseudouridine, 2-thio-pseudouridine,5-hydroxy-uridine (ho⁵U), 5-aminoallyl-uridine, 5-halo-uridine (e.g.,5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo⁵U),uridine 5-oxyacetic acid methyl ester (mcmo⁵U), 5-carboxymethyl-uridine(cm⁵U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine(chm⁵U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm⁵U),5-methoxycarbonylmethyl-uridine (mcm⁵U),5-methoxycarbonylmethyl-2-thio-uridine (mcm⁵s²U),5-aminomethyl-2-thio-uridine (nm⁵s²U), 5-methylaminomethyl-uridine(mnm⁵U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine(mnm⁵s²U), 5-methylaminomethyl-2-seleno-uridine (mnm⁵se²U),5-carbamoylmethyl-uridine (ncm⁵U), 5-carboxymethylaminomethyl-uridine(cmnm⁵U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm⁵s²U),5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine(τm⁵U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine(τm5s2U), 1-taurinomethyl-4-thio-pseudouridine), 5-methyl-2-thio-uridine(m⁵s²U), 1-methyl-4-thio-pseudouridine (m¹s⁴ψ),4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m³ψ),2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D),dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m⁵D),2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine,2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine,4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine,3-(3-amino-3-carboxypropyl)uridine (acp³U),1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp³ ψ),5-(isopentenylaminomethyl)uridine (inm⁵U),5-(isopentenylaminomethyl)-2-thio-uridine (inm⁵s²U), α-thio-uridine,2′-O-methyl-uridine (Um), 5,2′-O-dimethyl-uridine (m⁵Um),2′-O-methyl-pseudouridine (ψm), 2-thio-2′-O-methyl-uridine (s²Um),5-methoxycarbonylmethyl-2′-O-methyl-uridine (mcm⁵Um),5-carbamoylmethyl-2′-O-methyl-uridine (ncm⁵Um),5-carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm⁵Um),3,2′-O-dimethyl-uridine (m³Um),5-(isopentenylaminomethyl)-2′-O-methyl-uridine (inm⁵Um), 1-thio-uridine,deoxythymidine, 2′-F-ara-uridine, 2′-F-uridine, 2′-OH-ara-uridine,5-(2-carbomethoxyvinyl) uridine, and 5-[3-(1-E-propenylamino)uridine. Inone particularly preferred embodiment, the nucleoside comprising amodified nucleobase is pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ)or 5-methyl-uridine (m5U), in particular N1-methyl-pseudouridine.

In one embodiment, the replacement of one or more uridines with anucleoside comprising a modified nucleobase comprises a replacement ofat least 1%, at least 2%, at least 3%, at least 4%, at least 5%, atleast 10%, at least 25%, at least 50%, at least 75%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% of the uridines.

During synthesis of RNA (e.g., mRNA) by in vitro transcription (IVT)using T7 RNA polymerase significant amounts of aberrant products,including double-stranded RNA (dsRNA) are produced due to unconventionalactivity of the enzyme. dsRNA induces inflammatory cytokines andactivates effector enzymes leading to protein synthesis inhibition.dsRNA can be removed from RNA such as IVT RNA, for example, by ion-pairreversed phase HPLC using a non-porous or porous C-18polystyrene-divinylbenzene (PS-DVB) matrix. Alternatively, an enzymaticbased method using E. coli RNaseIII that specifically hydrolyzes dsRNAbut not ssRNA, thereby eliminating dsRNA contaminants from IVT RNApreparations can be used. Furthermore, dsRNA can be separated from ssRNAby using a cellulose material. In one embodiment, an RNA preparation iscontacted with a cellulose material and the ssRNA is separated from thecellulose material under conditions which allow binding of dsRNA to thecellulose material and do not allow binding of ssRNA to the cellulosematerial.

As the term is used herein, “remove” or “removal” refers to thecharacteristic of a population of first substances, such asnon-immunogenic RNA, being separated from the proximity of a populationof second substances, such as dsRNA, wherein the population of firstsubstances is not necessarily devoid of the second substance, and thepopulation of second substances is not necessarily devoid of the firstsubstance. However, a population of first substances characterized bythe removal of a population of second substances has a measurably lowercontent of second substances as compared to the non-separated mixture offirst and second substances.

In one embodiment, the removal of dsRNA from non-immunogenic RNAcomprises a removal of dsRNA such that less than 10%, less than 5%, lessthan 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, lessthan 0.3%, or less than 0.1% of the RNA in the non-immunogenic RNAcomposition is dsRNA. In one embodiment, the non-immunogenic RNA is freeor essentially free of dsRNA. In some embodiments, the non-immunogenicRNA composition comprises a purified preparation of single-strandednucleoside modified RNA. For example, in some embodiments, the purifiedpreparation of single-stranded nucleoside modified RNA is substantiallyfree of double stranded RNA (dsRNA). In some embodiments, the purifiedpreparation is at least 90%, at least 91%, at least 92%, at least 93%,at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, at least 99.5%, or at least 99.9% single stranded nucleosidemodified RNA, relative to all other nucleic acid molecules (DNA, dsRNA,etc.).

In one embodiment, the non-immunogenic RNA is translated in a cell moreefficiently than standard RNA with the same sequence. In one embodiment,translation is enhanced by a factor of 2-fold relative to its unmodifiedcounterpart. In one embodiment, translation is enhanced by a 3-foldfactor. In one embodiment, translation is enhanced by a 4-fold factor.In one embodiment, translation is enhanced by a 5-fold factor. In oneembodiment, translation is enhanced by a 6-fold factor. In oneembodiment, translation is enhanced by a 7-fold factor. In oneembodiment, translation is enhanced by an 8-fold factor. In oneembodiment, translation is enhanced by a 9-fold factor. In oneembodiment, translation is enhanced by a 10-fold factor. In oneembodiment, translation is enhanced by a 15-fold factor. In oneembodiment, translation is enhanced by a 20-fold factor. In oneembodiment, translation is enhanced by a 50-fold factor. In oneembodiment, translation is enhanced by a 100-fold factor. In oneembodiment, translation is enhanced by a 200-fold factor. In oneembodiment, translation is enhanced by a 500-fold factor. In oneembodiment, translation is enhanced by a 1000-fold factor. In oneembodiment, translation is enhanced by a 2000-fold factor. In oneembodiment, the factor is 10-1000-fold. In one embodiment, the factor is10-100-fold. In one embodiment, the factor is 10-200-fold. In oneembodiment, the factor is 10-300-fold. In one embodiment, the factor is10-500-fold. In one embodiment, the factor is 20-1000-fold. In oneembodiment, the factor is 30-1000-fold. In one embodiment, the factor is50-1000-fold. In one embodiment, the factor is 100-1000-fold. In oneembodiment, the factor is 200-1000-fold. In one embodiment, translationis enhanced by any other significant amount or range of amounts.

In one embodiment, the non-immunogenic RNA exhibits significantly lessinnate immunogenicity than standard RNA with the same sequence. In oneembodiment, the non-immunogenic RNA exhibits an innate immune responsethat is 2-fold less than its unmodified counterpart. In one embodiment,innate immunogenicity is reduced by a 3-fold factor. In one embodiment,innate immunogenicity is reduced by a 4-fold factor. In one embodiment,innate immunogenicity is reduced by a 5-fold factor. In one embodiment,innate immunogenicity is reduced by a 6-fold factor. In one embodiment,innate immunogenicity is reduced by a 7-fold factor. In one embodiment,innate immunogenicity is reduced by a 8-fold factor. In one embodiment,innate immunogenicity is reduced by a 9-fold factor. In one embodiment,innate immunogenicity is reduced by a 10-fold factor. In one embodiment,innate immunogenicity is reduced by a 15-fold factor. In one embodiment,innate immunogenicity is reduced by a 20-fold factor. In one embodiment,innate immunogenicity is reduced by a 50-fold factor. In one embodiment,innate immunogenicity is reduced by a 100-fold factor. In oneembodiment, innate immunogenicity is reduced by a 200-fold factor. Inone embodiment, innate immunogenicity is reduced by a 500-fold factor.In one embodiment, innate immunogenicity is reduced by a 1000-foldfactor. In one embodiment, innate immunogenicity is reduced by a2000-fold factor.

The term “exhibits significantly less innate immunogenicity” refers to adetectable decrease in innate immunogenicity. In one embodiment, theterm refers to a decrease such that an effective amount of thenon-immunogenic RNA can be administered without triggering a detectableinnate immune response. In one embodiment, the term refers to a decreasesuch that the non-immunogenic RNA can be repeatedly administered withouteliciting an innate immune response sufficient to detectably reduceproduction of the protein encoded by the non-immunogenic RNA. In oneembodiment, the decrease is such that the non-immunogenic RNA can berepeatedly administered without eliciting an innate immune responsesufficient to eliminate detectable production of the protein encoded bythe non-immunogenic RNA. “Immunogenicity” is the ability of a foreignsubstance, such as RNA, to provoke an immune response in the body of ahuman or other animal. The innate immune system is the component of theimmune system that is relatively unspecific and immediate. It is one oftwo main components of the vertebrate immune system, along with theadaptive immune system.

As used herein “endogenous” refers to any material from or producedinside an organism, cell, tissue or system.

As used herein, the term “exogenous” refers to any material introducedfrom or produced outside an organism, cell, tissue or system.

The term “expression” as used herein is defined as the transcriptionand/or translation of a particular nucleotide sequence.

As used herein, the terms “linked,” “fused”, or “fusion” are usedinterchangeably. These terms refer to the joining together of two ormore elements or components or domains.

Codon-Optimization/Increase in G/C Content

In some embodiment, the amino acid sequence comprising a SARS-CoV-2 Sprotein, an immunogenic variant thereof, or an immunogenic fragment ofthe SARS-CoV-2 S protein or the immunogenic variant thereof describedherein is encoded by a coding sequence which is codon-optimized and/orthe G/C content of which is increased compared to wild type codingsequence. This also includes embodiments, wherein one or more sequenceregions of the coding sequence are codon-optimized and/or increased inthe G/C content compared to the corresponding sequence regions of thewild type coding sequence. In one embodiment, the codon-optimizationand/or the increase in the G/C content preferably does not change thesequence of the encoded amino acid sequence.

The term “codon-optimized” refers to the alteration of codons in thecoding region of a nucleic acid molecule to reflect the typical codonusage of a host organism without preferably altering the amino acidsequence encoded by the nucleic acid molecule. Within the context of thepresent disclosure, coding regions are preferably codon-optimized foroptimal expression in a subject to be treated using the RNA moleculesdescribed herein. Codon-optimization is based on the finding that thetranslation efficiency is also determined by a different frequency inthe occurrence of tRNAs in cells. Thus, the sequence of RNA may bemodified such that codons for which frequently occurring tRNAs areavailable are inserted in place of “rare codons”.

In some embodiments of the present disclosure, the guanosine/cytosine(G/C) content of the coding region of the RNA described herein isincreased compared to the G/C content of the corresponding codingsequence of the wild type RNA, wherein the amino acid sequence encodedby the RNA is preferably not modified compared to the amino acidsequence encoded by the wild type RNA. This modification of the RNAsequence is based on the fact that the sequence of any RNA region to betranslated is important for efficient translation of that mRNA.Sequences having an increased G (guanosine)/C (cytosine) content aremore stable than sequences having an increased A (adenosine)/U (uracil)content. In respect to the fact that several codons code for one and thesame amino acid (so-called degeneration of the genetic code), the mostfavourable codons for the stability can be determined (so-calledalternative codon usage). Depending on the amino acid to be encoded bythe RNA, there are various possibilities for modification of the RNAsequence, compared to its wild type sequence. In particular, codonswhich contain A and/or U nucleotides can be modified by substitutingthese codons by other codons, which code for the same amino acids butcontain no A and/or U or contain a lower content of A and/or Unucleotides.

In various embodiments, the G/C content of the coding region of the RNAdescribed herein is increased by at least 10%, at least 20%, at least30%, at least 40%, at least 50%, at least 55%, or even more compared tothe G/C content of the coding region of the wild type RNA. In someembodiments, G/C content of a coding region is increased by about 10% toabout 60% (e.g., by about 20% to about 60%, about 30% to about 60%,about 40% to about 60%, about 50% to about 60%, or by about 51%, about52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%,about 59%, or about 60%) compared to the G/C content of the codingregion of the wild type RNA.

In some embodiments, RNA disclosed herein comprises a sequence disclosedherein (e.g., SEQ ID NO: 9), that has been modified to encode one ormore mutations characteristic of a SARS-CoV-2 variant (e.g., onesdescribed herein including but not limited to a BA.2 or a BA.4/5 Omicronvariant). In some embodiments, RNA can be modified to encode one or moremutations characteristic of a SARS-CoV-2 variant by making as fewnucleotide changes as possible. In some embodiments, RNA can be modifiedto encode one or more mutations that are characteristic of a SARS-CoV-2variant by introducing mutations that result in high codon-optimizationand/or increased G/C content.

In some embodiments, one or more mutations characteristic of aSARS-CoV-2 variant are introduced onto a full-length S protein (e.g., anS protein comprising SEQ ID NO: 1). In some embodiments one or moremutations characteristic of a SARS-CoV-2 variant are introduced onto afull-length S protein having one or more proline mutations that increasestability of a prefusion confirmation. For example, in some embodiments,proline substitutions are made at positions corresponding to positions986 and 987 of SEQ ID NO: 1. In some embodiments, at least 4 prolinesubstitutions are made. In some embodiments, at least four of suchproline mutations include mutations at positions corresponding toresidues 817, 892, 899, and 942 of SEQ ID NO: 1, e.g., as described inWO 2021243122 A2, the entire contents of which are incorporated hereinby reference in its entirety. In some embodiments, such a SARS-CoV-2 Sprotein comprising proline substitutions at positions corresponding toresidues 817, 892, 899, and 942 of SEQ ID NO: 1, may further compriseproline substitutions at positions corresponding to residues 986 and 987of SEQ ID NO: 1. In some embodiments, one or more mutationscharacteristic of a SARS-CoV-2 variant are introduced onto animmunogenic fragment of an S protein (e.g., the RBD of SEQ ID NO: 1).

Embodiments of Administered RNAs

In some embodiments, the present disclosure provides an RNA (e.g., mRNA)comprising an open reading frame encoding a polypeptide that comprisesat least a portion of a SARS-CoV-2 S protein. The RNA is suitable forintracellular expression of the polypeptide. In some embodiments, suchan encoded polypeptide comprises a sequence corresponding to thecomplete S protein. In some embodiments, such an encoded polypeptidedoes not comprise a sequence corresponding to the complete S protein. Insome embodiments, the encoded polypeptide comprises a sequence thatcorresponds to the receptor binding domain (RBD). In some embodiments,the encoded polypeptide comprises a sequence that corresponds to theRBD, and further comprises a trimerization domain (e.g., a trimerizationdomain as disclosed herein, such as a fibritin domain). In someembodiments an RBD comprises a signaling domain (e.g., a signalingdomain as disclosed herein). In some embodiments an RBD comprises atransmembrane domain (e.g., a transmembrane domain as disclosed herein).In some embodiments, an RBD comprises a signaling domain and atrimerization domain. In some embodiments, an RBD comprises a signalingdomain, a trimerization domain, and transmembrane domain.

In some embodiments, the encoded polypeptide comprises a sequence thatcorresponds to two receptor binding domains. In some embodiments, theencoded polypeptide comprises a sequence that corresponds to tworeceptor binding domains in tandem in an amino acid chain, e.g., asdisclosed in Dai, Lianpan, et al. “A universal design of betacoronavirusvaccines against COVID-19, MERS, and SARS,” Cell 182.3 (2020): 722-733,the contents of which are incorporated by reference herein in theirentirety.

In some embodiments, a SARS-CoV-2 S protein, or an immunogenic fragmentthereof comprises one or more mutations to alter or remove aglycosylation site, e.g., as described in WO2022221835A2,US20220323574A1, or WO2022195351A1.

In some embodiments, compositions or medical preparations describedherein comprise RNA encoding an amino acid sequence comprisingSARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenicfragment of the SARS-CoV-2 S protein or the immunogenic variant thereof.Likewise, methods described herein comprise administration of such RNA.

The active platform for use herein is based on an antigen-coding RNAvaccine to induce robust neutralising antibodies andaccompanying/concomitant T cell response to achieve protectiveimmunization with preferably minimal vaccine doses. The RNA administeredis preferably in-vitro transcribed RNA.

Three different RNA platforms are particularly preferred, namelynon-modified uridine containing mRNA (uRNA), nucleoside modified mRNA(modRNA) and self-amplifying RNA (saRNA). In one particularly preferredembodiment, the RNA is in vitro transcribed RNA. In some embodiments,uRNA is mRNA. In some embodiments, modRNA is mRNA.

In the following, embodiments of these three different RNA platforms aredescribed, wherein certain terms used when describing elements thereofhave the following meanings:

S1M2 protein/S1S2 RBD: Sequences encoding the respective antigen ofSARS-CoV-2.

nsP1, nsP2, nsP3, and nsP4: Wildtype sequences encoding the Venezuelanequine encephalitis virus (VEEV) RNA-dependent RNA polymerase replicaseand a subgenomic promotor plus conserved sequence elements supportingreplication and translation.

virUTR: Viral untranslated region encoding parts of the subgenomicpromotor as well as replication and translation supporting sequenceelements.

hAg-Kozak: 5′-UTR sequence of the human alpha-globin mRNA with anoptimized ‘Kozak sequence’ to increase translational efficiency.

Sec: Sec corresponds to a secretory signal peptide (sec), which guidestranslocation of the nascent polypeptide chain into the endoplasmaticreticulum. In some embodiments, such a secretory signal peptide includesthe intrinsic S1S2 secretory signal peptide. In some embodiments, such asecretory signal peptide is a secretory signal peptide from a non-S1S2protein. For example, an immunoglobulin secretory signal peptide (aa1-22), an HSV-1 gD signal peptide (MGGAAARLGAVILFVVIVGLHGVRSKY (SEQ IDNO: 105)), an HSV-2 gD signal peptide (MGRLTSGVGTAALLVVAVGLRVVCA (SEQ IDNO: 106)); a human SPARC signal peptide, a human insulin isoform 1signal peptide, a human albumin signal peptide, or any other signalpeptide described herein.

Glycine-serine linker (GS): Sequences coding for short linker peptidespredominantly consisting of the amino acids glycine (G) and serine (S),as commonly used for fusion proteins.

Fibritin: Partial sequence of T4 fibritin (foldon), used as artificialtrimerization domain.

TM: TM sequence corresponds to the transmembrane part of a protein. Atransmembrane domain can be N-terminal, C-terminal, or internal to anencoded polypeptide. A coding sequence of a transmembrane element istypically placed in frame (i.e., in the same reading frame), 5′, 3′, orinternal to coding sequences of sequences (e.g., sequences encodingpolypeptide(s)) with which it is to be linked. In some embodiments, atransmembrane domain comprises or is a transmembrane domain ofHemagglutinin (HA) of Influenza virus, Env of HIV-1, equine infectiousanaemia virus (EIAV), murine leukaemia virus (MLV), mouse mammary tumorvirus, G protein of vesicular stomatitis virus (VSV), Rabies virus, or aseven transmembrane domain receptor. In some embodiments, thetransmembrane part of a protein is from the S1S2 protein.

FI element: The 3′-UTR is a combination of two sequence elements derivedfrom the “amino terminal enhancer of split” (AES) mRNA (called F) andthe mitochondrial encoded 12S ribosomal RNA (called 1). These wereidentified by an ex vivo selection process for sequences that confer RNAstability and augment total protein expression.

A30L70: A poly(A)-tail measuring 110 nucleotides in length, consistingof a stretch of adenosine residues, followed by a 10 nucleotide linkersequence and another 70 adenosine residues designed to enhance RNAstability and translational efficiency in dendritic cells.

In some embodiments, vaccine RNA described herein may comprise, from 5′to 3′, one of the following structures:

-   -   Cap-5′-UTR-Vaccine Antigen-Encoding Sequence-3′-UTR-Poly(A)        or    -   Cap-hAg-Kozak-Vaccine Antigen-Encoding Sequence-FI-A30L70.

In some embodiments, a vaccine antigen described herein may comprise afull-length S protein or an immunogenic fragment thereof (e.g., RBD). Insome embodiments where a vaccine antigen comprises a full-length Sprotein, its secretory signal peptide and/or transmembrane domain may bereplaced by a heterologous secretory signal peptide (e.g., as describedherein) and/or a heterologous transmembrane domain (e.g., as describedherein).

In some embodiments, a vaccine antigen described herein may comprise,from N-terminus to C-terminus, one of the following structures:

-   -   Signal Sequence-RBD-Trimerization Domain        or    -   Signal Sequence-RBD-Trimerization Domain-Transmembrane Domain.

RBD and Trimerization Domain may be separated by a linker, in particulara GS linker such as a linker having the amino acid sequence GSPGSGSGS(SEQ ID NO: 134). Trimerization Domain and Transmembrane Domain may beseparated by a linker, in particular a GS linker such as a linker havingthe amino acid sequence GSGSGS (SEQ ID NO: 135).

Signal Sequence may be a signal sequence as described herein. RBD may bea RBD domain as described herein. Trimerization Domain may be atrimerization domain as described herein. Transmembrane Domain may be atransmembrane domain as described herein.

In one embodiment,

-   -   Signal sequence comprises the amino acid sequence of amino acids        1 to 16 or 1 to 19 of SEQ ID NO: 1 or the amino acid sequence of        amino acids 1 to 22 of SEQ ID NO: 31, or an amino acid sequence        having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%        identity to this amino acid sequence,    -   RBD comprises the amino acid sequence of amino acids 327 to 528        of SEQ ID NO: 1, or an amino acid sequence having at least 99%,        98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to this amino acid        sequence,    -   Trimerization Domain comprises the amino acid sequence of amino        acids 3 to 29 of SEQ ID NO: 10 or the amino acid sequence of SEQ        ID NO: 10, or an amino acid sequence having at least 99%, 98%,        97%, 96%, 95%, 90%, 85%, or 80% identity to this amino acid        sequence; and    -   Transmembrane Domain comprises the amino acid sequence of amino        acids 1207 to 1254 of SEQ ID NO: 1, or an amino acid sequence        having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%        identity to this amino acid sequence.

In one embodiment,

-   -   Signal sequence comprises the amino acid sequence of amino acids        1 to 16 or 1 to 19 of SEQ ID NO: 1 or the amino acid sequence of        amino acids 1 to 22 of SEQ ID NO: 31,    -   RBD comprises the amino acid sequence of amino acids 327 to 528        of SEQ ID NO: 1,    -   Trimerization Domain comprises the amino acid sequence of amino        acids 3 to 29 of SEQ ID NO: 10 or the amino acid sequence of SEQ        ID NO: 10; and    -   Transmembrane Domain comprises the amino acid sequence of amino        acids 1207 to 1254 of SEQ ID NO: 1.    -   In some embodiments, an RNA polynucleotide comprising a sequence        encoding a vaccine antigen (e.g., a SARS-CoV-2 S protein, an        immunogenic variant thereof, or an immunogenic fragment of the        SARS-CoV-2 S protein or the immunogenic variant thereof) or        comprising an open reading frame encoding a vaccine antigen        (e.g., a SARS-CoV-2 S protein, an immunogenic variant thereof,        or an immunogenic fragment of the SARS-CoV-2 S protein or the        immunogenic variant thereof) such as the nucleotide sequence of        SEQ ID NO: 50 or the nucleotide sequence of SEQ ID NO: 53, a        variant or fragment thereof, further comprises a 5′ cap, e.g., a        5′ cap comprising a Cap1 structure, a 5′ UTR sequence, e.g., a        5′ UTR sequence comprising the nucleotide sequence of SEQ ID NO:        12, a 3′ UTR sequence, e.g., a 3′ UTR sequence comprising the        nucleotide sequence of SEQ ID NO: 13, and polyA sequence, e.g.,        a polyA sequence comprising the nucleotide sequence of SEQ ID        NO: 14. In some embodiments, RNA is formulated in a composition        comprising        ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate),        cholesterol, distearoylphosphatidylcholine, and        (2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide).

RNA described herein or RNA encoding the vaccine antigen describedherein may be non-modified uridine containing RNA (uRNA), nucleosidemodified RNA (modRNA) or self-amplifying RNA (saRNA). In someembodiments, uRNA is mRNA. In some embodiments, modRNA is mRNA. In oneembodiment, RNA described herein or RNA encoding the vaccine antigendescribed herein is nucleoside modified RNA (modRNA).

Variant Specific Vaccines

In some embodiments, RNA disclosed herein encodes an S proteincomprising one or more mutations that are characteristic of a SARS-CoV-2variant. In some embodiments, RNA encodes a SARS-CoV-2 S proteincomprising one or more mutations characteristic of an Alpha variant. Insome embodiments, RNA encodes a SARS-CoV-2 S protein comprising one ormore mutations characteristic of a Beta variant. In some embodiments,RNA encodes a SARS-CoV-2 S protein comprising one or more mutationscharacteristic of a Delta variant. In some embodiments, RNA encodes aSARS-CoV-2 S protein comprising one or more mutations characteristic ofan Omicron variant (e.g., an S protein comprising one or more mutationscharacteristic of a BA.1, BA.2, or BA.4/5 Omicron variant). In someembodiments, RNA encodes a SARS-CoV-2 S protein comprising one or moremutations characteristic of an BA.1 Omicron variant. In someembodiments, RNA encodes a SARS-CoV-2 S protein comprising one or moremutations characteristic of an BA.2 Omicron variant. In someembodiments, RNA encodes a SARS-CoV-2 S protein comprising one or moremutations characteristic of an BA.2.12.1 Omicron variant. In someembodiments, RNA encodes a SARS-CoV-2 S protein comprising one or moremutations characteristic of a BA.3 Omicron variant. In some embodiments,RNA encodes a SARS-CoV-2 S protein comprising one or more mutationscharacteristic of a BA.4 or BA.5 Omicron variant.

Non-Modified Uridine RNA (uRNA)

In some embodiments, a non-modified uridine RNA is a messenger RNA. Insome embodiments, the active principle of non-modified messenger RNAdrug substance is a single-stranded mRNA that is translated uponentering a cell. In addition to the sequence encoding the coronavirusvaccine antigen (i.e. open reading frame), each uRNA preferably containscommon structural elements optimized for maximal efficacy of the RNAwith respect to stability and translational efficiency (including, e.g.,5′-cap, 5′-UTR, 3′-UTR, poly(A)-tail as described herein). The preferred5′ cap structure is beta-S-ARCA(D1) (m₂ ^(7,2′-O)GppSpG). The preferred5′-UTR and 3′-UTR comprise the nucleotide sequence of SEQ ID NO: 12 andthe nucleotide sequence of SEQ ID NO: 13, respectively. The preferredpoly(A)-tail comprises the sequence of SEQ ID NO: 14.

Different embodiments of this platform are as follows:

RBL063.1 (SEQ ID NO: 15; SEQ ID NO: 7) Structurebeta-S-ARCA(D1)-hAg-Kozak- S1S2-PP-FI-A30L70 EncodedViral spike protein (S1S2 protein) antigenof the SARS-COV-2 (S1S2 full-length protein, sequence variant)RBL063.2 (SEQ ID NO: 16; SEQ ID NO: 7) Structurebeta-S-ARCA(D1)-hAg-Kozak- S1S2-PP-FI-A30L70 EncodedViral spike protein (S1S2 protein) antigenof the SARS-COV-2 (S1S2 full-length protein, sequence variant)BNT162a1; RBL063.3 (SEQ ID NO: 17; SEQ ID NO: 5) Structurebeta-S-ARCA(D1)-hAg-Kozak- RBD-GS-Fibritin-FI-A30L70 EncodedViral spike protein (S protein) antigenof the SARS-COV-2 (partial sequence, Receptor Binding Domain(RBD) of S1S2 protein)

FIG. 3 schematizes the general structure of the antigen-encoding RNAs.

Nucleoside Modified RNA (modRNA)

In some embodiments, nucleoside modified RNA is mRNA. In someembodiments, the active principle of nucleoside modified RNA (modRNA)drug substance is a single-stranded RNA (e.g., mRNA) that can betranslated upon entering a cell. In addition to a sequence encoding acoronavirus vaccine antigen (i.e., open reading frame), each modRNAcontains common structural elements optimized for maximal efficacy ofthe RNA as the uRNA (5′-cap, 5′-UTR, 3′-UTR, poly(A)-tail). Compared touRNA, modRNA comprises at least one nucleotide modification (e.g., asdescribed herein). In some embodiments, modRNA contains1-methyl-pseudouridine instead of uridine. The preferred 5′ capstructure is m₂ ^(7,3′-O)Gppp(m₁ ^(2′-O))ApG. The preferred 5′-UTR and3′-UTR comprise the nucleotide sequence of SEQ ID NO: 12 and thenucleotide sequence of SEQ ID NO: 13, respectively. The preferredpoly(A)-tail comprises the sequence of SEQ ID NO: 14. An additionalpurification step is applied for modRNA to reduce dsRNA contaminantsgenerated during the in vitro transcription reaction.

Different embodiment of this platform are as follows:

BNT162b2; RBP020.1 (SEQ ID NO: 19; SEQ ID NO: 7) Structure m₂^(7,3′-O)Gppp(m₁ ^(2′-O))ApG)-hAg- Kozak-S1S2-PP-FI-A30L70 EncodedViral spike protein (S1S2 protein) of antigenthe SARS-COV-2 (S1S2 full-length protein, sequence variant)BNT162b2; RBP020.2 (SEQ ID NO: 20; SEQ ID NO: 7) Structure m₂^(7,3′-O)Gppp(m₁ ^(2′-O))ApG)-hAg- Kozak-5152-PP-FI-A30L70 EncodedViral spike protein (S1S2 protein) of antigenthe SARS-COV-2 (S1S2 full-length protein, sequence variant)BNT162b1; RBP020.3 (SEQ ID NO: 21; SEQ ID NO: 5) Structure m₂^(7,3′-O)Gppp(m₁ ^(2′-O))ApG)-hAg- Kozak-RBD-GS-Fibritin-FI-A30L70Encoded Viral spike protein (S1S2 protein) of antigenthe SARS-Cov-2 (partial sequence, Receptor Binding Domain (RBD) of S1S2protein fused to fibritin)

FIG. 4 schematizes the general structure of the antigen-encoding RNAs.

BNT162b3c (SEQ ID NO: 29; SEQ ID NO: 30) Structure m₂ ^(7,3′-O)Gppp(m₁^(2′-O))ApG-hAg-Kozak- RBD-GS-Fibritin-GS-TM-FI-A30L70 EncodedViral spike protein (S1S2 protein) of the SARS-Cov-2 (partial sequence,antigen Receptor Binding Domain (RBD) of S1S2 protein fused toFibritin fused to Transmembrane Domain (TM) of S1S2 protein);intrinsic S1S2 protein secretory signal peptide(aa 1-19) at the N-terminus of the antigen sequenceBNT162b3d (SEQ ID NO: 31; SEQ ID NO: 32) Structure m₂ ^(7,3′-O)Gppp(m₁^(2′-O))ApG-hAg-Kozak-RBD-GS- Fibritin-GS-TM-FI-A30L70 EncodedViral spike protein (S1S2 protein) of the SARS-Cov-2 (partial sequence,antigenReceptor Binding Domain (RBD) of S1S2 protein fused to Fibritin fused toTransmembrane Domain (TM) of S1S2 protein); immunoglobulin secretorysignal peptide (aa 1-22) at the N-terminus of the antigen sequence.BNT162b2-Beta variant; RBP020.11 (SEQ ID NO: 57; SEQ ID NO: 55)Structure m₂ ^(7,3′-O)Gppp(m₁ ^(2′-O))ApG)-hAg-Kozak-S1S2-PP-FI-A30L70EncodedViral spike protein (S1S2 protein) of the SARS-COV-2 (S1S2 full-lengthantigenprotein, sequence variant), comprising mutations characteristic of theBeta variant of SARS-COV-2BNT162b2-Alpha variant; RBP020.14 (SEQ ID NO: 60; SEQ ID NO: 58)Structure m₂ ^(7,3′-O)Gppp(m₁ ^(2′-O))ApG)-hAg-Kozak-S1S2-PP-FI-A30L70EncodedViral spike protein (S1S2 protein) of the SARS-COV-2 (S1S2 full-lengthantigenprotein, sequence variant), comprising mutations characteristic of theAlpha variant of SARS-COV-2BNT162b2-Delta variant; RBP020.16 (SEQ ID NO: 63a; SEQ ID NO: 61)Structure m₂ ^(7,3′-O)Gppp(m₁ ^(2′-O)}ApG)-hAg-Kozak-S1S2-PP-FI-A30L70EncodedViral spike protein (S1S2 protein) of the SARS-COV-2 (S1S2 full-lengthantigenprotein, sequence variant), comprising mutations characteristic of theDelta variant of SARS-COV-2

Nucleotide Sequence of RBP020.11 (Beta-Specific Vaccine)

Nucleotide sequence (SEQ ID NO: 153) is shown with individual sequenceelements as indicated in bold letters. In addition, the sequence of thetranslated protein (SEQ ID NO:55) is shown in italic letters below thecoding nucleotide sequence (*=stop codon). Red text indicates pointmutations in the nucleotide and amino acid sequences.

        10         20         30         40         50  53AGAATAAACT AGTATTCTTC TGGTCCCCAC AGACTCAGAG AGAACCCGCC ACC                          hAg-Kozak        63         73         83         93        103        313ATGTTCGTGT TCCTGGTGCT GCRGCCTCTG GTGTCCAGCC AGTGTGTGAA CTTCACCACC  H  F  V   F  L  V   L  L  P  L   V  S  S   Q  C  V   N  F  T  T                          S Protein mut       123        133        143        153        163        173AGAACACAGC TGCCTCCAGC CTACACCAAC AGCTTTACCA GAGGCGTGTA CTACCCCGAC  R  T  Q   L  P  P   A  Y  T  N   S  F  T   R  G  V   Y  Y  P  D                          S Protein mut       183        193        203        213        223        233AAGGTGTTCA GATCCAGCGT GCTGCACTCT ACCCAGGACC TGTTCCTGCC TTTCTTCAGC  K  V  F   R  S  S   C  L  H  S   T  Q  D   L  F  L   F  F  F  S                          S Protein mut       243        253        263        273        283        293AACGTGACCT GGTTCCACGC CATCCACGTG TCCGGCACCA ATGGCACCAA GAGATTCGCC  N  V  T   W  F  H   A  I  H  V   S  G  T   N  G  T   K  R  F  A                          S Protein mut       303        313        323        333        343        353AACCCCGTGC TGCCCTTCAA CGACGGGGTG TACTTTGCCA GCACCGAGAA GTCCAACATC  N  P  V   L  P  F   N  D  G  V   Y  F  A   S  T  E   K  S  N  I                          S Protein mut       383        373        383        383        403        433ATCAGAGGCT GGATCTTCGG CACCACACTG GACAGCAAGA CCCAGAGCCT GCTGATCGTG  I  R  G   W  I  F   G  T  T  L   D  S  K   T  Q  S   L  L  I  V                          S Protein mut       423        433        443        453        463        473AACAACGCCA CCAACGTGGT CATCAAAGTG TGCGAGTTCC AGTTCTGCAA CGACCCCTTC  N  N  A   T  N  V   V  I  K  V   C  E  F   Q  F  C   N  D  P  F                          S Protein mut       483        493        503        513        523        533CTGGGCGTCT ACTACCACAA GAACAACAAG AGCTGGATGG AAAGCGAGTT CCGGGTGTAC  L  G  V   Y  Y  H   K  N  N  K   S  W  M   E  S  E   F  R  V  Y                          S Protein mut       543        553        563        573        583        593AGCAGCGCCA ACAACTGCAC CTTCGAGTAC GTGTCCCAGC CTTTCCTGAT GGACCTGGAA  S  S  A   N  N  C   T  F  E  Y   V  S  Q   F  F  L   M  D  L  E                          S Protein mut       603        613        623        633        643        653GGCAAGCAGG GCAACTTCAA GAACCTGCGC GAGTTCGTGT TTAACAACAT CGACGGCTAC  G  K  Q   K  N  F   K  N  L  R   E  F  V   F  K  N   T  D  G  Y                          S Protein mut       663        673        683        693        703        713TTCAAGATCT ACAGCAAGCA CACCCCTATC AACCTCGTGC GGGGCCTGCC TCAGGGCTTC  F  K  I   Y  S  K   H  T  P  I   N  L  V   R  G  L   F  Q  G  F                          S Protein mut       723        733        743        753        763        773TCTGCTCTGG AACCCCTGGT GGATCTGCCC ATCGGCATCA ACATCACCCG GTTTCAGACA  S  A  L   E  P  L   V  D  L  P   I  G  I   N  I  T   R  F  Q  T                          S Protein mut       783        793        803        813        823        833CTGCACATCA GCTACCTGAC ACCTGGCGAT AGCAGCAGCG GATGGACAGC TGGTGCCGCC  L  H  Y  S  Y  L  T  P  G  D  S  D  D  G  W  T  A  G  A  A                          S Protein mut       843        353        853        873        883        893GCTTAGTATG TGGGCTACCT GCAGCCTAGA ACCTTCCTGC TGAAGTACAA CGAGAACGGC  A  Y  Y   V  G  Y   L  Q  P  R   T  F  L   L  K  Y   N  E  N  G                          S Protein mut       903        913        923        933        943        953ACCATCACCG ACGCCGTGGA TTGTGCTCTG GATCCTCTGA GCGAGACAAA GTGCACCCTG  T  I  T   D  A  V   D  C  A  L   D  F  L   D  E  T   K  C  T  L                          S Protein mut       963        973        983        993       1003       1013AAGTCCTTCA CCGTGGAAAA GGGCATCTAC CAGACCAGCA ACTTCCGGGT GCAGCCCACC  K  G  F   T  V  E   K  G  I  Y   Q  T  S   N  F  R   V  Q  P  T                          S Protein mut      1023       1033       1043       1053       1063       1073GAATCCATCG TGCGGTTCCC CAATATCACC AATCTGTGCC CCTTCGGCGA GGTGTTCAAT  E  S  I   V  R  F   P  N  I  T   N  L  C   P  F  G   E  V  F  N                          S Protein mut      1033       1093       1103       1113       1123       1133GCCACGAGAT TCGCCTCTGT GTACGCCTGG AACCGGAAGC GGATCAGCAA TTGCGTGGCC  A  T  R   F  A  S   V  Y  A  W   N  R  K   R  I  S   N  C  V  A                          S Protein mut      1143       1153       1163       1173       1183       1193GACTACTCCG TGCTGTACAA CTCCGCCAGC TTCAGCACCT TCAAGTGCTA CGGCGTGTCC  D  Y  S   V  L  Y   N  S  A  S   F  S  F   F  K  C   Y  G  V  S                          S Protein mut      1203       1213       1223       1233       2243       1253CCTACCAAGC IGAACGACCT GTGCTTCACA AACGTGTACG CCGACAGCTT CGTGATCCGG  P  T  K   L  N  D   L  C  F  T   N  V  Y   A  D  S   F  V  I  R                          S Protein mut      1268       1273       1283       1293       1303       1313GGAGATGAAG TGCGGCAGAT TGCCCCTGGA CAGACAGGCA ACATCGCCGA CTACAACTAC  G  D  E   V  R  Q   I  A  F  G   Q  T  G   N  I  A   D  Y  N  Y                          S Protein mut      1323       1333       1343       1353       1363       1373AAGCTGCCCG ACGACTTCAC CGGCTGTGTG ATTGCCTGGA ACAGCAACAA CCTGGACTCC  K  L  F   D  D  F   T  G  C  V   I  A  W   N  S  N   N  L  D  S                          S Protein mut      1383       1393       1403       1413       1423       1433AAAGTCGGCG GCAACTACAA TTACCTGTAC CGGCTGTTCC GGAAGTCCAA TCTGAAGCCC  K  V  G   G  N  Y   N  Y  L  Y   R  L  F   R  K  S   N  L  K  P                          S Protein mut      1443       1453       1463       1473       1483       1493TTCGAGCGGG ACATCTCCAC CGAGATCTAT CAGGCCGCCA GCACCCCTTG TAACGGCGTG  F  E  R   D  I  S   T  E  I  Y   Q  A  G   S  T  P   C  N  G  V                          S Protein mut      1503       1513       1523       1533       1543       1553AAGGGCTTCA ACTGCTACTT CCCACTGCAG TCCTACGGCT TTCAGCCCAC ATACGGCGTG  K  G  F   N  C  Y   E  P  L  Q   S  Y  G   F  Q  P   T  T  G  V                          S Protein mut      1563       1573       1583       1593       1603       1613GGCTATCAGC CCTACAGAGT GGTGGTGCTG AGCTTCGAAC TGCTGCATGC CCCTGCCACA  G  Y  Q   P  Y  R   V  V  V  L   S  F  E   L  L  N   A  P  A  T                          S Protein mut      1623       1633       1643       1653       1673GTGTGCGGCC CTAAGAAAAG CACCAATCTC CTGAAGAACA AATGCGTGAA CTTCAACTTC  V  C  G   P  K  K   S  T  N  L   V  K  N   K  C  V   N  F  N  F                          S Protein mut      1683       1693       1703       1713       1723       1733AACGGCCTCA CCGGCACCGG CGTGCTGACA GAGAGCAACA AGAAGTTCCT GCCATTCCAG  N  G  L   T  G  T   G  V  L  T   E  G  N   K  K  F   L  P  F  Q                          S Protein mut      1743       1753       1763       1773       1783       1793CAGTTTGGCC GGGATATCGC CGATACCACA GACGCCGTTA GAGATCCCCA GACACTGGAA  Q  F  G   R  D  I   A  D  T  T   D  A  V   R  D  F   Q  T  L  E                          S Protein mut      1803       1813       1823       1833       1843       1853ATCCTGGACA TCACCCCTTG CAGCTTCGGC GGAGTGTCTG TGATCACCCC TGGCACCAAC  I  L  D   I  R  P   C  S  F  G   G  V  S   V  I  F   P  G  T  N                          S Protein mut      1863       1873       1883       1893       1903       1913ACCAGCAATC AGGTGGCAGT GCTGTACCAG CGCGTGAACT GTACCGAAGT GCCCGTGGCC  T  S  N   Q  V  A   V  L  Y  Q   G  V  N   C  T  E   V  P  V  A                          S Protein mut      1923       1933       1943       1953       1963       1973ATTCACGCCG ATCAGCTGAC ACCTACATGG CGGGTGTACT CCACCGGCAG CAATGTGTTT  I  H  A   D  Q  L   T  P  T  W   R  V  Y   S  T  G   S  N  V  F                          S Protein mut      1983       1993       2003       2013       2023       2033CAGACCAGAG CCGGCTGTCT GATCGGAGCC GAGCACGTGA ACAATAGCTA CGAGTGCGAC  Q  T  R   A  G  C   L  I  G  A   E  K  V   N  N  S   Y  E  C  D                          S Protein mut      2043       2053       2063       2073       2083       2093ATCCCCATCG GCGCTGGAAT CTGCGCCAGC TACCAGACAG AGACAAACAG CCCTCGGAGA  I  P  I   G  A  G   I  C  A  S   Y  Q  T   Q  T  N   S  F  R  R                          S Protein mut      2103       2113       2123       2133       2143       2153GCCAGAAGCG TGGCCAGCCA GAGCATCATT GCCTACACAA TGTCTCTGGG CGTCGAGAAC  A  R  S   V  A  S   Q  S  I  I   A  Y  T   M  S  L   G  V  E  N                          S Protein mut      2163       2173       2183       2193       2203       2213AGCGTGGCCT ACTCCAACAA CTCTATCGCT ATCCCCACCA ACTTCACCAT CAGCGTGACC  S  V  A   Y  S  N   N  S  I  A   I  F  T   N  F  T   I  S  V  T                          S Protein mut      2223       2233       2243       2253       2263       2273ACAGAGATCC TGCCTGTGTC CATGACCAAG ACCAGCGTGA ACTGCACCAT GTACATCTGC  T  E  I   L  F  V   S  N  T  K   T  S  V   D  C  T   N  Y  I  C                          S Protein mut      2283       2293       2303       2313       2323       2333GGCGATTCCA CCGAGAGCTC CAACCTGCTG CTGCAGTACG GCAGCTTCTG CACCCAGCTG  G  D  S   T  E  C   S  N  L  L   L  Q  T   G  S  F   C  T  Q  L                          S Protein mut      2343       2353       2363       2373       2383       2393AATAGAGCCC TGACAGGGAT CGCCGTGGAA CAGGACAAGA ACACCCAAGA GGTGTTCGCC  N  R  A  L  T  G  I  A  V  E  Q  D  K  N  T  Q  S  V  F  A                          S Protein mut      2403       2413       2423       2433       2443       2453CAAGTGAAGC AGATCTACAA GACCCCTCCT ATCAAGGACT TCGGCGGCTT CAATTTCAGC  Q  V  K   Q  I  Y   K  T  T  P   I  K  D   F  G  G   F  N  F  S                          S Protein mut      2463       2473       2483       2493       2503       2513CAGATTCTGC CCGATCCTAG CAACCCCAGC AAGCGGAGCT TCATCGAGGA CCTGCTGTTC  Q  I  L   F  D  F   S  K  F  D   K  R  D   F  I  E   D  L  L  F                          S Protein mut      2523       2533       2543       2553       2563       2573AACAAAGTGA CACTGGCCGA CGCCGGCTTC ATCAAGCAGT ATGGCGATTG TCTGGGCGAC  N  K  V   T  L  A   D  A  G  F   I  K  Q   Y  G  D   C  L  G  DS Protein mout      2583       2593       2603       2613       2623       2633ATTGCGGCCA GGGATCTGAT TTGCGCCCAG AAGTTTAACG GACTGACAGT GCTGCCTCCT  I  A  A   R  D  L   I  C  A  Q   K  F  N   G  L  T   V  L  F  F                          S Protein mut      2643       2653       2663       2673       2683       2693CTGCTGACCG ATGAGATGAT CGCCCAGTAC ACATCTGCCC TGCTGGCCGG CACAATCACA  L  L  T   D  E  M   I  A  Q  Y   T  S  A   L  L  A   G  T  I  T                          S Protein mut      2703       2713       2723       2733       2743       2753AGCGGCTGGA CATTTGGAGC AGGCGCCGCT CTGCAGATCC CCTTTGCTAT GCAGATGGCC  S  G  W   T  G  A   A  G  A  A   L  Q  I   P  F  A   N  Q  N  A                          S Protein mut      2763       2773       2783       2793       2803       2813TACCGGTTCA ACGGCATCGG AGTGACCCAG AATGTGCTGT ACGAGAACCA GAAGCTGATC  Y  R  F   N  G  I   G  V  T  Q   N  V  L   Y  E  N   Q  K  L  I                          S Protein mut      2823       2833       2843       2853       2863       2873GCCAACCAGT TCAACAGCGC CATCGGCAAG ATCCAGGACA GCCTGAGCAG CACAGCAAGC  A  N  Q   F  N  S   A  I  G  K   I  Q  D   S  L  S   S  T  A  S                          S Protein mut      2883       2893       2903       2913       2923       2933GCCCTGGGAA AGCTGCAGGA CGTGGTCAAC CAGAATGGCC AGGCACTGAA CACCCTGGTC  A  L  G   K  L  Q   D  V  V  N   Q  N  A   Q  A  L   N  T  L  V                          S Protein mut      2943       2953       2963       2973       2983       2993AAGCAGCTGT CCTCCAACTT CGGCGCCATC AGCTCTGTGC TGAACGATAT CCTGAGCACA  K  Q  L   S  S  N   F  G  A  I   S  S  V   L  N  D   I  L  S  R                          S Protein mut      3003       3013       3023       3033       3043       3053CTGGACCCTC CTGAGGCCGA GGTGCAGATC GACAGACTGA TCACAGGCAG ACTGCAGAGC  L  D  P   P  E  A   E  V  Q  I   D  R  L   I  T  G   R  L  Q  S                          S Protein mut      3063       3073       3083       3093       3103       3113CTCCAGACAT ACGTGACCCA GCAGCTGATC AGAGCCGCCG AGATTAGAGC CTCTGCCAAT  L  Q  T   Y  V  T   Q  Q  L  I   R  A  A   E  I  R   A  S  A  N                          S Protein mut      3123       3133       3143       3153       3163       3173CTGGCCGCCA CCAAGATGTC TGAGTGTGTG CTGGGCCAGA GCAAGAGAGT GGACTTTTGC  L  A  A   T  K  N   S  E  C  V   L  G  Q   S  K  R   V  D  F  C                          S Protein mut      3183       3193       3203       3213       3223       3233GGCAAGGGCT ACCACCTGAT GAGCTTCCCT CAGTCTGCCC CTCACGGCGT GGTGTTTCTG  G  K  G   Y  H  L   M  S  F  P   Q  S  A   P  H  G   V  V  F  L                          S Protein mut      3243       3253       3263       3273       3283       3293CACGTGACAT ATGTGCCCGC TCAAGAGAAG AATTTCACCA CCGCTCCAGC CATCTGCCAC  H  V  T   Y  V  P   A  Q  E  K   N  F  T   T  A  P   A  I  C  H                          S Protein mut      3303       3313       3323       3333       3343       3353GACGGCAAAG CCCACTTTCC TAGAGAAGGC GTGTTCGTGT CCAACGGCAC CCATTGGTTC  D  G  K   A  H  F   P  R  E  G   V  F  V   S  N  G   T  H  W  F                          S Protein mut      3363       3373       3383       3393       3403       3413GTGACACAGG GGAACTTCTA CGAGCCCCAG ATCATCACCA CCGACAACAC CTTCGTGTCT  V  T  Q   R  N  F   Y  E  P  Q   I  I  T   T  D  N   T  F  V  S                          S Protein mut      3423       3433       3443       3453       3463       3473GGCAACTGCG ACGTCGTGAT CGGCATTGTG AACAATACCG TGTACGACCC TCTGCAGCCC  G  N  C   D  V  V   I  G  I  V   N  N  T   V  Y  D   P  L  Q  F                          S Protein mut      3483       3493       3503       3513       3523       3533GAGCTGGACA GCTTCAAAGA GGAACTGGAC AAGTACTTTA AGAACCACAC AAGCCCCGAC  E  L  D   S  F  K   E  E  L  D   K  Y  F   K  N  K   T  S  F  D                          S Protein mut      3543       3553       3563       3573       3583       3593GTGGACCTGG GCGATATCAG CGGAATCAAT GCCAGCGTCG TGAACATCCA GAAAGAGATC  V  D  L   G  D  I   S  G  I  N   A  S  V   V  N  I   Q  R  E  I                          S Protein mut      3603       3613       3623       3633       3643       3653GACCGGCTGA ACGAGGTGGC CAAGAATCTG AACGAGAGCC TGATCGACCT GCAAGAACTG  D  R  L   N  E  V   A  K  N  L   N  E  S   L  I  D   L  Q  E  L                          S Protein mut      3663       3673       3683       3893       3703       3713GGGAAGTACG AGCAGTACAT CAAGTGGCCC TGGTACATCT GGCTGGGCTT TATCGCCGGA  G  K  Y   E  Q  Y   I  K  F  P   W  Y  I   W  L  G   F  I  A  G                          S Protein mut      3723       3733       3743       3753       3763       3773CTGATTGCCA TCGTGATGGT CACAATCATG CTGTGTTGCA TGACCAGCTG CTGTAGCTGC  L  I  A   I  V  N   V  T  I  N   L  C  C   N  T  S   C  C  S  C                          S Protein mut      3783       3793       3803       3813       3823       3833CTGAAGGGCT GTTGTAGCTG TGGCAGCTGC TGCAAGTTCG ACGAGGACGA TTCTGAGCCC  L  K  G   C  C  S   S  G  S  C   C  K  F   D  E  D   D  S  E  P                          S Protein mut      3843       3853       3863    3670GTGCTGAAGG GCGTGAAACT GCACTACACA TGATGAC  V  L  K   G  V  K   L  H  Y  T   *  *                          S Protein mut      3880       3890       3900       3910       3920       3930TCGAGCTGGT ACTGCATGCA CGCAATGCTA GCTGCCCCTT TCCCGTCCTG GGTACCCCGA                          FI Element      3940       3850       3960       3970       3980       3990GTCTCCCCCG ACCTCGGGTC CCAGGTATGC TCCCACCTCC ACCTGCCCCA CTCACCACCT                          FI Element      4000       4010       4020       4030       4040       4050CTGCTAGTTC CAGACACCTC CCAAGCACGC AGCAATGCAG CTCAAAACGC TTAGCCTAGC                          FI Element      4060       4070       4080       4090       4100       4110CACACCCCCA CGGGAAACAG CAGTGATTAA CCTTTAGCAA TAAACGAAAG TTTAACTAAG                          FI Element      4120       4130       4140       4150       4160       4164CTATACTAAC CCCAGGGTTG GTCAATTTCG TGCCAGCCAC ACCCTGGAGC TAGC                          FI Element      4174       4184       4194       4204       4214       4224AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA GCATATGACT AAAAAAAAAA AAAAAAAAAA                          A30L70      4234       4244       4254       4264       4274AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA                          A30L70

Sequences of RBP020.11 are also shown in Table 3.

TABLE 3 Sequences of RBP020.11 (a Beta-specific RNA vaccine) SEQ ID NO.Brief Description Sequence   55 Amino acidMFVFLVLLPLVSSQCVNFTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFH sequence of RNA-AIHVSGTNGTKRFANPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEencoded SARS-CoV-2FQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDS proteinGYFRIYSKHTPINDVRGLPQGFSALEPLVDLPIGINITRFQTLHISYLTPGDSSSGWTAGAAAYYVfrom a BetaGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITvariantNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADS(with PROFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFEmutations atRDISTEIYQAGSTPCNGVKGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTpositionsNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVcorresponding toITPGTNTSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDK986P andIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGVENSVAYSNNSIAIPTNFTISVTTEILPVV987P of SEQ IDSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKNO: 1; i.e., PRODFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLmutations atPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQEpositions 983 andNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDPPEAEVQI984 of SEQ IDDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGNO: 55)VVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT**  56 RNA sequenceauguucgugu uccuggugcu gcugccucug guguccagcc agugugugaa cuucaccaccencoding a SARS-agaacacagc ugccuccagc cuacaccaac agcuuuacca gaggcgugua cuaccccgacCoV-2 S proteinaagguguuca gauccagcgu gcugcacucu acccaggacc uguuccugcs uuucuucagcfrom a Betaaacgugaccu gguuccacgc cauccacgug uccggcacca auggcaccaa gagauucgccvariantaaccccgugc ugcccuucaa cgacggggug uacuuugcca gcaccgagas guccaacaucaucagaggcu ggaucuucgg caccacacug gacagcaaga cccagagccu gcugaucgugaacaacgcca ccaacguggu caucaaagug ugcgaguucc aguucugcas cgaccccuuccugggcgucu acuaccacaa gaacaacaag agcuggaugg aaagcgaguu ccggguguacagcagcgcca acaacugcac cuucgaguac gugucccagc cuuuccugau ggaccuggaaggcaagcagg gcaacuucaa gaaccugcgc gaguucgugu uusagaacau cgacggcuacuucaagaucu acagcaagca caccccuauc aaccucgugc ggggccugcc ucagggcuucucugcucugg aaccccuggu ggaucugccc aucggcauca acaucacccg guuucagacacugcacauca gcuaccugac accuggcgau agcagcagcg gauggacagc uggugccgccgcuuacuaug ugggcuaccu gcagccuaga accuuccugc ugaaguacaa cgagaacggcaccaucaccg acgccgugga uugugcucug gauccucuga gcgagacaaa gugcacccugaaguccuuca ccguggaaaa gcgcaucuac cagaccagca acuuccgggu gcagcccaccgaauccaucg ugcgguuccc caauaucacc aaucugugcc ccuucggcga gguguucaaugccaccagau ucgccucugu guacgccugg aaccggaagc ggaucagcaa uugcguggccgacuacuccg ugcuguacaa cuccgccagc uucagcaccu ucaagugcua cggcguguccccuaccaagc ugaacgaccu gugcuucaca aacguguacg ccgacagcuu cgugauccggggagaugaag ugcggcagau ugccccugga cagacaggca acaucgccga cuacaacuacaagcugcccg acgacuucac cggcugugug auugccugga acagcaacaa ccuggacuccaaagucggcg gcaacuacaa uuaccuguac cggcuguucc ggaaguccaa ucugaagcccuucgagcggg acaucuccac cgagaucuau caggccggca gcaccccuug uaacggcgugaagggcuuca acugcuacuu cccacugcag uccuacggcu uucagcccac auacggcgugggcuaucagc ccuacagagu gguggugcug agcuucgaac ugcugcaugc cccugccacagugugcggcc cuaagaaaag caccaaucuc gugaagaaca aaugcgugaa cuucaacuucaacggccuga ccggcaccgg cgugcugaca gagagcaaca agaaguuccu gccauuccagcaguuuggcr gggauaucgc cgauaccaca gacgccguua gagaucccca gacacuggaaauccuggaca ucaccccuug cagcuucggc ggagugucug ugaucacccc uggcaccaacaccagcaauc agguggcagu gcuguaccag ggcgugaacu guaccgaagu gcccguggccauucacgccg aucagcugac accuacaugg cggguguacu ccaccggcag caauguguuucagaccagag ccggcugucu gaucggagcc gagcacguga acaauagcua cgagugcgacauccccaucg gcgcuggaau cugcgccagc uaccagacac agacaaacag cccucggagagccagaagcg uggccagcca gagcaucauu gccuacacaa ugucucuggg cgucgagaacagcguggccu acuccaacaa cucuaucgcu auccccacca acuucaccau cagcgugaccacagagaucc ugccuguguc caugaccaag accagcgugg acugcaccau guacaucugcggcgauucca ccgagugcuc caaccugcug cugcaguacg gcagcuucug cacccagcugaauagagccc ugacagggau ccccguggaa caggacaaga acacccaaga gguguucgcccaagugaagc agaucuacaa gaccccuccu aucaaggacu ucggcggcuu caauuucagccagauucugc ccgauccuag caagcccagc aagcggagcu ucaucgagga ccugcuguucaacaaaguga cacuggccga ccccggcuuc aucaagcagu auggcgauug ucugggcgacauugccgcca gggaucugau uugcgcccag aaguuuaacg gacugacagu gcugccuccucugcugaccg augagaugau cgcccaquac acaucugccc ugcuggccgg cacaaucacaagcggcugga cauuuggagc aggcgccgcu cugcagaucc ccuuugcuau gcagauggccuaccgguuca acggcaucgg agugacccag aaugugcugu acgagaacca gaagcugaucgccaaccagu ucaacagcgc caucggcaag auccaggaca gccugagcag cacagcaagcgcccugggaa agcugcagga ccuggucaac cagaaugccc aggcacugaa cacccuggucaagcagcugu ccuccaacuu cggcgccauc agcucugugc ugaacgauau ccugagcagacuggacccuc cugaggccga ggugcagauc gacagacuga ucacaggcag acugcagagccuccagacau acgugaccca gcagcugauc agagccgccg agauuagagc cucugccaaucuggccgcca ccaagauguc ugagugugug cugggccaga gcaagagagu ggacuuuugcggcaagggcu accaccugau gagcuucccu cagucugccc cucacggcgu gguguuucugcacgugacau augugcccgc ucaagagaag aauuucacca ccgcuccagc caucugccadgacggcaaag cccacuuucc uagagaaggc guguucgugu ccaacggcac ccauugguucgugacacagc ggaacuucua cgagccccag aucaucacca ccgacaacac cuucgugucuggcaacugcg acqucgugau ccgcauugug aacaauaccg uquacgaccc ucugcagcccgagcuggaca gcuucaaaga ggaacuggac aaguacuuua agaaccacac aagccccgacguggaccugg gcgauaucag ccgaaucaau gccagcgucg ugaacaucca gaaagagaucgaccggcuga acgagguggc caagaaucug aacgagagcc ugaucgaccu gcaagaacuggggaaguacg agcaguacau caaguggccc ugguacaucu ggcugggcuu uaucgccggacugauugcca ucgugauggu cacaaucaug cuguguugca ugaccagcug cuguagcugccugaagggcu guuguagcug uggcagcugc ugcaaguucg acgaggacga uucugagcccgugcugaagg gcgugaaacu gcacuacaca ugaugac 152 DNA sequenceatgttcgtgt tcctggtgct gctgcctctg gtgtccagcc agtgtgtgaa cttcaccaccencoding a SARS-agaacacagc tgcctccagc ctacaccaac agctttacca gaggcgtgta ctaccccgacCoV-2 S proteinaaggtqttca gatccagcgt gctgcactct acccaggacc tcttcctgcc tttcttcagcfrom a Betaaacgtgacct ggttccacgc catccacqtq tccggcacca atqqcaccaa gagattcqccvariantaaccccgtgc tgcccttcaa cgacggggtg tactttgcca gcaccgagaa gtccaacatcatcagaggct ggatcttcgg caccacactg gacagcaaga cccagagcct gctgatcgtgaacaacgcca ccaacgtggt catcaaagtg tgcgagttcc agttctgcaa cgaccccttcctgggcgtct actaccacaa gaacaacaag agctggatgg aaagcgagtt ccgggtgtacagcagcgcca acaactgcac cttcgagtac gigtcccagc ctttcctgat ggacctggaaggcaagcagg gcaacttcaa gaacctgcgc gagttcgtgt ttaagaacat cgacggctacttcaagatct acagcaagca cacccctatc aacctcgtgc ggggcctgcc tcagggcttctctgctctgg aacccctggt ggatctgccc atcggcatca acatcacccg gtttcagacactgcacatca gctacctgac acctggcgat agcagcagcg gatggacagc tcgtgccgccgcttactatg tgggctacct gcagcctaga accttcctgc tgaagtacaa cgagaacggcaccatcaccg acgccgtgga ttgtgctctg gatcctctga gcgagacaaa gtgcaccctgaagtccttca ccgtggaaaa gggcatctac cagaccagca acttccgggt gcagcccaccgaatccatcg tgcggttccc caatatcacc aatctgtgcc ccttcggcgc ggtgttcaatgccaccagat tcgcctctgt gtacgcctgg aaccggaagc ggatcagcaa ttgcgtggccgactactccg tgctgtacaa ctccgccagc ttcagcacct tcaagtgcta cggcgtgtcccctaccaagc tgaacgacct gtgcttcaca aacgtgtacg ccgacagctt cgtgatccggggagatgaag tgcggcagat tgcccctgga cagacaggca acatcgccga ctacaactacaagctgcccg acgacttcac ccgctgtgtg attgcctgga acagcaacaa cctggactccaaagtcggcg gcaactacaa ttacctgtac ccgctgttcc ggaagtccaa tctgaagcccttcgagcggg acatctccac cgagatctat caggccggca gcaccccttg taacggcgtgaagggcttca actgctactt cccactgcag tcctacggct ttcagcccac atacggcgtgggctatcagc cctacagagt ggtggtgctg agcttcgaac tgctgcatgc ccctgccacagtgtgcggcc ctaagaaaag caccaatctc gtgaagaaca aatgcgtgaa cttcaacttcaacggcctga ccggcaccgg cgtgctgaca gagagcaaca agaagttcct gccattccagcagtttggcc gggatatcgc cgataccaca gacgccgtta gagatcccca gacactggaaatcctggaca tcaccccttg cagcttcggc ggagtgtctg tgatcacccc tggcaccaacaccagcaatc aggtggcagt gctgtaccag ggcgtgaact gtaccgaagt gcccgtggccattcacgccg atcagctgac acctacatgg cgggtqtact ccaccggcag caatgtgtttcagaccagag ccggctgtct gatcggagcc gagcacgtga acaatagcta cgagtgcgacatccccatcg gcgctggaat ctgcgccagc taccagacac agacaaacag ccctcggagagccagaagcg tcgccagcca gagcatcatt gcctacacaa tctctctggg cgtcgagaacagcgtggcct actccaacaa ctctatcgct atccccacca acttcaccat cagcgtgaccacagagatcc tgcctgtgtc catgaccaag accagcgtyg actgcaccat gtacatctgcggcgattcca ccgagtgctc caacctgctg ctgcagtacg gcagcttctg cacccagctgaatagagccc tgacagggat cgccgtggaa caggacaaga acacccaaga ggtgttcgcccaagtgaagc agatctacaa gacccctcct atcaaggact tcggcggctt caatttcagccagattctgc ccgatcctag caagcccagc aagcggagct tcatcgagga cctgctgttcaacaaagtga cactggccga ccccggcttc atcaagcagt atggcgattg tctgggcgacattgccgcca gggatctgat ttgcgcccag aagtttaacg gactgacagt gctgcctcctctgctgaccg atgagatgat cgcccagtac acatctgccc tgctggccgg cacaatcacaagcggctgga catttggagc aggcgccgct ctgcagatcc cctttgctat gcagatggcctaccggttca acggcatcgg agtgacccag aatgtgctgt acgagaacca gaagctgatcgccaaccagt tcaacagcgc catcggcaag atccaggaca gcctgagcag cacagcaagcgccctgggaa agctgcagga cgtggtcaac cagaatgccc aggcactgaa caccctggtcaagcagctgt cctccaactt cggcgccatc agctctgtgc tgaacgatat cctgagcagactggaccctc ctgaggccga ggtgcagatc gacagactga tcacaggcag actgcagagcctccagacat acqtgaccca gcagctgatc agagccgccg agattagagc ctctgccaatctggccgcca ccaagatgtc tgagtgtgtg ctgggccaga gcaagagagt ggacttttgcggcaagggct accacctgat gagcttccct cagtctgccc ctcacggcgt ggtgtttctgcacgtgacat atgtgcccgc tcaagagaag aatttcacca ccgctccagc catctgccacgacggcaaag cccactttcc tagagaaggc gtgttcgtgt ccaacggcac ccattggttcgtgacacagc ggaacttcta cgagccccag atcatcacca ccgacaacac cttcgtgtctggcaactgcg acgtcgtgat cggcattgtg aacaataccg tgtacgaccc tctgcagcccgagctggaca gcttcaaaga ggaactggac aagtacttta agaaccacac aagccccgacgtggacctgg gcgatatcag cggaatcaat gccagcgtcg tgaacatcca gaaagagatcgaccggctga acgaggtggc caagaatctg aacgagagcc tgatcgacct gcaagaactggggaagtacg agcagtacat caagtggccc tcgtacatct ggctgggctt tatcgccggactgattgcca tcgtgatggt cacaatcaty ctgtgttgca tgaccagctg ctgtagctgcctgaagggct gttgtagctg tygcagctgc tgcaagttcg acgaggacga ttctgagcccgtgctgaagg gcgtgaaact gcactacaca tgatgac 57 Full lengthagaauaaacu aguauucuuc ugguccccac agacucagag agaacccgcc accRNA sequence ofauguucgugu uccuggugcu cgacggggug guguccagcc agugugugaa cuucaccaccRBP020.11agaacacagc ugccuccagc gcugccucug agcuuuacca gaggcgugua cuaccccgacaagguguuca gauccagcgu cuacaccaac acccaggacc uguuccugcc uuucuucagcaacgugaccu gguuccacgc gcugcacucu uccggcacca auggcaccaa gagauucgccaaccccgugc ugcccuucaa cauccacgug uacuuugcca gcaccgagaa guccaacaucaucagaggcu ggaucuucgg caccacacug gacagcaaga cccagagccu gcugaucgugaacaacgcca ccaacguggu caucaaagug ugcgaguucc aguucugcaa cgaccccuuccugggcgucu acuaccacaa gaacaacaag agcuggaugg aaagcgaguu ccggguguacagcagcgcca acaacugcac cuucgaguac gugucccagc cuuuccugau ggaccuggaaggcaagcagg gcaacuucaa gaaccugcgc gaguucgugu uusagaacau cgacggcuacuucaagaucu acagcaagca caccccuauc aaccucgugc ggggccugcc ucagggcuucucugcucugg aaccccuggu ggaucugccc aucggcauca acaucacccg guuucagacacugcacauca gcuaccugac accuggcgau agcagcagcg gauggacagc uggugccgccgcuuacuaug ugggcuaccu gcagccuaga accuuccugc ugaaguacaa cgagaacggcaccaucaccg acgccgugga uugugcucug gauccucuga gcgagacaaa gugcacccugaaguccuuca ccguggaaaa gggcaucuac cagaccagca acuuccgggu gcagcccaccgaauccaucg ugcgguuccc caauaucacc aaucugugcc ccuucggcga gguguucaaugccaccagau ucgccucugu guacgccugg aaccggaagc ggaucagcaa uugcguggccgacuacuccg ugcuguacaa cuccgccagc uucagcaccu ucaagugcua cggcguguccccuaccaagc ugaacgaccu gugcuucaca aacguguacg ccgacagcuu cgugauccggggagaugaag ugcggcagau ugccccugga cagacaggca acaucgccga cuacaacuacaagcugcccg acgacuucac ccgcugugug auugccugga acagcaacaa ccuggacuccaaaguccgcg gcaacuacaa uuaccuquac ccgcuguucc ggaaguccaa ucugaagcccuucgagcggg acaucuccac cgagaucuau caggccggca gcaccccuug uaacggcgugaagggcuuca acugcuacuu cccacugcag uccuacggcu uucagcccac auacggcgugggcuaucagc ccuacagagu gguggugcug agcuucgaac ugcugcaugc cccugccacagugugcggcc cuaagaaaag caccaaucuc gugaagaaca aaugcgugas cuucaacuucaacggccuga ccggcaccgg cgugcugaca gagagcaaca agaaguuccu gccauuccagcaguuuggcc gggauaucgc cgauaccaca gacgccguua gagaucccca gacacuggaaauccuggaca ucaccccuug cagcuucggc ggagugucug ugaucacccc uggcaccaacaccagcaauc agguggcagu gcuguaccag ggcgugaacu guaccgaagu gcccguggcccagaccagag ccggcugucu gaucggagcc gagcacguga acaauagcua cgagugcgacauccccaucg gcgcuggaau cugcgccagc uaccagacac agacaaacag cccucggagagccagaagcg uggccagcca gagcaucauu gccuacacaa ugucucuggg cgucgagaacagcguggccu acuccaacaa cucuaucgcu auccccacca acuucaccau cagcgugaccacagagaucc ugccuguguc caugacccag accagcgugg acugcaccau guacaucugcggcgauucca ccgagugcuc caaccugcug cugcaguacg gcagcuucug cacccagcugaauagagccc ugacagggau ccccguggaa caggacaaga acacccaaga gguguucgcccaagugaagc agaucuacaa gaccccuccu aucaaggacu ucggcggcuu caauuucagccagauucugc ccgauccuag caagcccagc aagcggagcu ucaucgagga ccugcuguucaacaaaguga cacuggccga cgccggcuuc aucaagcagu auggcgauug ucugggcgacauugccgcca gggaucugau uugcgcccag aaguuuaacg gacugacagu gcugccuccucugcugaccg augagaugau cgcccaguac acaucugccc ugcuggccgg cacaaucacaagcggcugga cauuuggagc aggcgccgcu cugcagaucc ccuuugcuau gcagauggccuaccgguuca acggcaucgg agugacccag aaugugcugu acgagaacca gaagcugaucgccaaccagu ucaacagcgc caucggcaag auccaggaca gccugagcag cacagcaagcgcccugggaa agcugcagga cguggucaac cagaaugccc aggcacugaa cacccuggucaagcagcugu ccuccaacuu cggcgccauc agcucugugc ugaacgauau ccugagcagacuggacccuc cugaggccga ggugcagauc gacagacuga ucacaggcag acugcagagccuccagacau acgugaccca gcagcugauc agagccgccg agauuagagc cucugccaauggcaagggcu accaccugau gagcuucccu cagucugccc cucacggcgu gguguuucugcacgugacau augugcccgc ucaagagaag aauuucacca ccgcuccagc caucugccacgacggcaaag cccacuuucc uagagaaggc guguucgugu ccaacggcac ccauugguucgugacacagc ggaacuucua cgagccccag aucaucacca ccgacaacac cuucgugucuggcaacugcg acgucgugau cggcauugug aacaauaccg uguacgaccc ucugcagcccgagcuggaca gcuucaaaga ggaacuggac aaguacuuua agaaccacac aagccccgacguggaccugg gcgauaucag cggaaucaau gccagcgucg ugaacaucca gaaagagaucgaccggcuga acgagguggc caagaaucug aacgagagcc ugaucgaccu gcaagaacuggggaaguacg agcaguacau caaguggccc ugguacaucu ggcugggcuu uaucgccggacugauugcca ucgugauggu cacaaucaug cuguguugca ugaccagcug cuguagcugccugaagggcu guuguagcug uggcagcugc ugcaaguucg acgaggacga uucugagcccgugcugaagg gcgugaaacu gcacuacaca ugaugacucgagcuggu acugcaugca cgcaaugcua gcugccccuu ucccguccug gguaccccgagucucccccg accucggguc ccagguaugc ucccaccucc accugcccca cucaccaccucugcuaguuc cagacaccuc ccaagcacgc agcaaugcag cucaaaacgc uuagccuagccacaccccca ccggaaacag cagugauuaa ccuuuagcaa uaaacgaaag uuuaacuaagcuauacuaac cccaggguug gucaauuucg ugccagccac acccuggagc uagcaaaaaaaaaa aaaaaaaaaa Aaaaaaaaaa Gcauaugacu aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 153 Full lengthagaataaact agtattcttc tcgtccccac agactcagag agaacccgcc accDNA sequence ofatgttcgtgt tcctggtgct gctgcctctg gtgtccagcc agtgtgtgaa cttcaccaccRBP020.11agaacacagc tccctccagc ctacaccaac agctttacca gaggcgtgta ctaccccgacaaggtgttca gatccagcgt gctgcactct acccaggacc tcttcctgcc tttcttcagcaacgtgacct ggttccacgc catccacgtg tccggcacca atggcaccaa gagattcgccaaccccgtgc tgcccttcaa cgacggggtg tactttgcca gcaccgagaa gtccaacatcatcagaggct ggatcttcgg caccacactg gacagcaaga cccagagcct gctgatcgtgaacaacgcca ccaacgtggt catcaaagtg tgcgagttcc agttctgcaa cgaccccttcctgggcgtct actaccacaa gaacaacaag agctggatgg aaagcgagtt ccgggtgtacagcagcgcca acaactgcac cttcgagtac gtgtcccagc ctttcctgat ggacctggaaggcaagcagg gcaacttcaa gaacctgcgc gagttcgtgt ttaagaacat cgacggctacttcaagatct acagcaagca cacccctatc aacctcgtgc ggggcctgcc tcagggcttctctgctctgg aacccctggt ggatctgccc atcggcatca acatcacccg gtttcagacactgcacatca gctacctgac acctggcgat agcagcagcg gatggacagc tggtgccgccgcttactatg tgggctacct gcagcctaga accttcctgc tgaagtacaa cgagaacggcaccatcaccg acgccgtgga ttgtgctctg gatcctctga gcgagacaaa gtgcaccctgaagtccttca ccgtcgaaaa gggcatctac cagaccagca acttccgggt gcagcccaccgaatccatcg tgcggttccc caatatcacc aatctgtgcc ccttcggcga ggtgttcaatgccaccagat tcgcctctgt gtacgcctgg aaccggaagc ggatcagcaa ttgcgtggccgactactccg tgctgtacaa ctccgccagc ttcagcacct tcaagtgcta cggcgtgtcccctaccaagc tgaacgacct gtgcttcaca aacgtgtacg ccgacagctt cgtgatccggggagatgaag tgcggcagat tgcccctgga cagacaggca acatcgccgc ctacaactacaagctgcccg acgacttcac cggctgtgtg attgcctgga acagcaacaa cctggactccaaagtcggcg gcaactacaa ttacctgtac cggctgttcc ggaagtccaa tctgaagcccttcgagcggg acatctccac cgagatctat caggccggca gcaccccttg taacggcgtgaagggcttca actgctactt cccactgcag tcctacggct ttcagcccac atacggcgtgggctatcagc cctacagagt gctggtgctg agcttcgaac tgctgcatgc ccctgccacagtgtgcggcc ctaagaaaag caccaatctc gtgaagaaca aatgcgtgaa cttcaacttcaacggcctga ccggcaccgg cgtgctgaca gagagcaaca agaagttcct gccattccagcagtttggcc gggatatcgc cgataccaca gacgccgtta gagatcccca gacactggaaatcctggaca tcaccccttg cagcttcggc ggagtgtcty tgatcacccc tygcaccaacaccagcaatc aggtygcagt gctgtaccag ggcgtgaact gtaccgaagt gcccgtggccattcacgccg atcagctgac acctacatgg cgggtgtact ccaccggcag caatgtgtttcagaccagag ccggctgtct gatcggagcc gagcacgtga acaatagcta cgagtgcgacatccccatcg gcgctggaat ctgcgccagc taccagacac agacaaacag ccctcggagagccagaagcg tcgccagcca gagcatcatt gcctacacaa tctctctggg cgtcgagaacagcgtggcct actccaacaa ctctatcgct atccccacca acttcaccat cagcgtgaccacagagatcc tgcctgtgtc catgaccaag accagcgtgg actgcaccat gtacatctgcggcgattcca ccgagtgctc caacctgcty ctgcagtacg gcagcttctg cacccagctgaatagagccc tgacagggat cgccgtggaa caggacaaga acacccaaga ggtgttcgcccaagtgaagc agatctacaa gacccctcct atcaaggact tcggcggctt caatttcagccagattctgc ccgatcctag caagcccagc aagcggagct tcatcgagga cctgctgttcaacaaagtga cactggccga cgccggcttc atcaagcagt atggcgattg tctgggcgacattgccgcca gggatctgat ttgcgcccag aagtttaacg gactgacagt gctgcctcctctgctgaccg atgagatgat cccccagtac acatctgccc tgctggccgg cacaatcacaagcggctgga catttggagc aggcgccgct ctgcagatcc cctttgctat gcagatggcctaccggttca acggcatcgg agtgacccag aatgtgctgt acgagaacca gaagctgatcgccaaccagt tcaacagcgc catcggcaag atccaggaca gcctgagcag cacagcaagcgccctgggaa agctgcagga cgtggtcaac cagaatgccc aggcactgaa caccctggtcaagcagctgt cctccaactt cggcgccatc agctctgtgc tgaacgatat cctgagcagactggaccctc ctgaggccga ggtgcagatc gacagactga tcacaggcag actgcagagcctccagacat acgtgaccca gcagctgatc agagccgccg agattagagc ctctgccaatctggccgcca ccaagatgtc tgagtgtgtg ctgggccaga gcaagagagt ggacttttgcggcaagggct accacctgat gagcttccct cagtctgccc ctcacggcgt ggtgtttctgcacgtgacat atgtgcccgc tcaagagaag aatttcacca ccgctccagc catctgccacgacggcaaag cccactttcc tagagaaggc gtgttcgtgt ccaacggcac ccattggttcgtgacacagc ggaacttcta cgagccccag atcatcacca ccgacaacac cttcgtgtctggcaactgcg acgtcgtgat cggcattgtg aacaataccg tctacgaccc tctgcagcccgagctggaca gcttcaaaga ggaactggac aagtacttta agaaccacac aagccccgacgtggacctgg gcgatatcag cggaatcaat gccagcgtcg tgaacatcca gaaagagatcgaccggctga acgaggtggc caagaatctg aacgagagcc tgatcgacct gcaagaactggggaagtacg agcagtacat caagtggccc tggtacatct ggctgggctt tatcgccggactgattgcca tcgtgatggt cacaatcatg ctgtgttgca tgaccagctg ctgtagctgcctgaagggct gttgtagctg tcgcagctgc tccaagttcg acgaggacga ttctgagcccgtgctgaagg gcgtgaaact gcactacaca tgatgactcgagctggt actgcatgca cgcaatgcta gctgcccctt tcccgtcctg ggtaccccgagtctcccccg acctcgggtc ccaggtatgc tcccacctcc acctgcccca ctcaccacctctgctagttc cagacacctc ccaagcacgc agcaatgcag ctcaaaacgc ttagcctagccacaccccca ccqqaaacag cagtgattaa cctttagcaa taaacgaaag tttaactaagctatactaac cccagggttg gtcaatttcg tgccagccac accctggagc tagcaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa gcatatcact aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 

Nucleotide Sequence of RBP020.14 (Alpha-Specific Vaccine)

Nucleotide sequence (SEQ ID NO: 60) is shown with individual sequenceelements as indicated in bold letters. In addition, the sequence of thetranslated protein (SEQ ID NO: 58) is shown in italic letters below thecoding nucleotide sequence (*=stop codon). Red text indicates pointmutations in both the nucleotide and amino acid sequences.

        10         20         30         40         50  53AGAAUAAACU AGUAUUCUUC UGGUCCCCAC AGACUCAGAG AGAACCCGCC ACC                         hAg-Kozak        63         73         83         93        103        113AUGUUCGUGU UCCUGGUGCU GCUGCCUCUG GUGUCCAGCC AGUGUGUGAA CCUGACCACC  M  F  V   F  L  V   L  L  P  L   V  S  S   Q  C  V   N  L  T  T                          S protein mut4       123        133        143        153        163        173AGAACACAGC UGCCUCCAGC CUACACCAAC AGCUUUACCA GAGGCGUGUA CUACCCCGAC  R  T  Q   L  P  P   A  Y  T  N   S  F  T   R  G  V   Y  Y  P  D                          S protein mut3       183        193        203        213        223        233AAGGUGUUCA GAUCCAGCGU GCUGCACUCU ACCCAGGACC UGUUCCUGCC UUUCUUCAGC  K  V  F   R  S  S   V  L  H  S   T  Q  D   L  F  L   P  F  F  S                          S protein mut3       243        253        263        273        283        293AACGUGACCU GGUUCCACGC CAUCUCCGGC ACCAAUGGCA CCAAGAGAUU CGACAACCCC  N  V  T   W  F  H   A  I  S  G   T  N  G   T  K  R   F  D  N  P                          S protein mut3       303        313        323        333        343        353GUGCUGCCCU UCAACGACGG GGUGUACUUU GCCAGCACCG AGAAGUCCAA CAUCAUCAGA  V  L  P   F  N  D   G  V  Y  F   A  S  T   E  K  S   N  I  I  R                          S protein mut3       363        373        383        393        403        413GGCUGGAUCU UCGGCACCAC ACUGGACAGC AAGACCCAGA GCCUGCUGAU CGUGAACAAC  G  W  I   F  G  T   T  L  D  S   K  T  Q   S  L  L   I  V  N  N                          S protein mut3       423        433        443        453        463        473GCCACCAACG UGGUCAUCAA AGUGUGCGAG UUCCAGUUCU GCAACGACCC CUUCCUGGGC  A  T  N   V  V  I   K  V  C  E   F  Q  F   C  N  D   P  F  L  G                          S protein mut3       483        493        503        513        523        533GUCUACCACA AGAACAACAA GAGCUGGAUG GAAAGCGAGU UCCGGGUGUA CAGCAGCGCC  V  Y  H   K  N  N   K  S  W  M   E  S  E   F  R  V   Y  S  S  A                          S protein mut3       543        553        563        573        583        593AACAACUGCA CCUUCGAGUA CGUGUCCCAG CCUUUCCUGA UGGACCUGGA AGGCAAGCAG  N  N  C   T  F  E   Y  V  S  Q   P  F  L   M  D  L   E  G  K  Q                          S protein mut3       603        613        623        633        643        653GGCAACUUCA AGAACCUGCG CGAGUUCGUG UUUAAGAACA UCGACGGCUA CUUCAAGAUC  G  N  F   K  N  L   R  E  F  V   F  K  N   I  D  G   Y  F  K  I                          S protein mut3       663        673        683        693        703        713UACAGCAAGC ACACCCCUAU CAACCUCGUG CGGGAUCUGC CUCAGGGCUU CUCUGCUCUG  Y  S  K   H  T  P   I  N  L  V   R  D  L   P  Q  G   F  S  A  L                          S protein mut3       723        733        743        753        763        773GAACCCCUGG UGGAUCUGCC CAUCGGCAUC AACAUCACCC GGUUUCAGAC ACUGCUGGCC  E  P  L   V  D  L   P  I  G  I   N  I  T   R  F  Q   T  L  L  A                          S protein mut3       783        793        803        813        823        833CUGCACAGAA GCUACCUGAC ACCUGGCGAU AGCAGCAGCG GAUGGACAGC UGGUGCCGCC  L  H  R   S  Y  L   T  P  G  D   S  S  S   G  W  T   A  G  A  A                          S protein mut3       843        853        863        873        883        893GCUUACUAUG UGGGCUACCU GCAGCCUAGA ACCUUCCUGC UGAAGUACAA CGAGAACGGC  A  Y  Y   V  G  Y   L  Q  P  R   T  F  L   L  K  Y   N  E  N  G                          S protein mut3       903        913        923        933        943        953ACCAUCACCG ACGCCGUGGA UUGUGCUCUG GAUCCUCUGA GCGAGACAAA GUGCACCCUG  T  I  T   D  A  V   D  C  A  L   D  P  L   S  E  T   K  C  T  L                          S protein mut3       963        973        983        993       1003       1013AAGUCCUUCA CCGUGGAAAA GGGCAUCUAC CAGACCAGCA ACUUCCGGGU GCAGCCCACC  K  S  F   T  V  E   K  G  I  Y   Q  T  S   N  F  R   V  Q  P  T                          S protein mut3      1023       1033       1043       1053       1063       1073GAAUCCAUCG UGCGGUUCCC CAAUAUCACC AAUCUGUGCC CCUUCGGCGA GGUGUUCAAU  E  S  I   V  R  F   P  N  I  T   N  L  C   P  F  G   E  V  F  N                          S protein mut3      1083       1093       1103       1113       1123       1133GCCACCAGAU UCGCCUCUGU GUACGCCUGG AACCGGAAGC GGAUCAGCAA UUGCGUGGCC  A  T  R   F  A  S   V  Y  A  W   N  R  K   R  I  S   N  C  V  A                          S protein mut3      1143       1153       1163       1173       1183       1193GACUACUCCG UGCUGUACAA CUCCGCCAGC UUCAGCACCU UCAAGUGCUA CGGCGUGUCC  D  Y  S   V  L  Y   N  S  A  S   F  S  T   F  K  C   Y  G  V  S                          S protein mut3      1203       1213       1223       1233       1243       1253CCUACCAAGC UGAACGACCU GUGCUUCACA AACGUGUACG CCGACAGCUU CGUGAUCCGG  P  T  K   L  N  D   L  C  F  T   N  V  Y   A  D  S   F  V  I  R                          S protein mut3      1263       1273       1283       1293       1303       1313GGAGAUGAAG UGCGGCAGAU UGCCCCUGGA CAGACAGGCA AGAUCGCCGA CUACAACUAC  G  D  E   V  R  Q   I  A  P  G   Q  T  G   K  I  A   D  Y  N  Y                          S protein mut3      1323       1333       1343       1353       1363       1373AAGCUGCCCG ACGACUUCAC CGGCUGUGUG AUUGCCUGGA ACAGCAACAA CCUGGACUCC  K  L  P   D  D  F   T  G  C  V   I  A  W   N  S  N   N  L  D  S                          S protein mut3      1383       1393       1403       1413       1423       1433AAAGUCGGCG GCAACUACAA UUACCUGUAC CGGCUGUUCC GGAAGUCCAA UCUGAAGCCC  K  V  G   G  N  Y   N  Y  L  Y   R  L  F   R  K  S   N  L  K  P                          S protein mut3      1443       1453       1463       1473       1483       1493UUCGAGCGGG ACAUCUCCAC CGAGAUCUAU CAGGCCGGCA GCACCCCUUG UAACGGCGUG  F  E  R   D  I  S   T  E  I  Y   Q  A  G   S  T  P   C  N  G  V                          S protein mut3      1503       1513       1523       1533       1543       1553GAAGGCUUCA ACUGCUACUU CCCACUGCAG UCCUACGGCU UUCAGCCCAC AUACGGCGUG  E  G  F   N  C  Y   F  P  L  Q   S  Y  G   F  Q  P   T  Y  G  V                          S protein mut3      1563       1573       1583       1593       1603       1613GGCUAUCAGC CCUACAGAGU GGUGGUGCUG AGCUUCGAAC UGCUGCAUGC CCCUGCCACA  G  Y  Q   P  Y  R   V  V  V  L   S  F  E   L  L  H   A  P  A  T                          S protein mut3      1623       1633       1643       1653       1663       1673GUGUGCGGCC CUAAGAAAAG CACCAAUCUC GUGAAGAACA AAUGCGUGAA CUUCAACUUC  V  C  G   P  K  K   S  T  N  L   V  K  N   K  C  V   N  F  N  F                          S protein mut3      1683       1693       1703       1713       1723       1733AACGGCCUGA CCGGCACCGG CGUGCUGACA GAGAGCAACA AGAAGUUCCU GCCAUUCCAG  N  G  L   T  G  T   G  V  L  T   E  S  N   K  K  F   L  P  F  Q                          S protein mut3      1743       1753       1763       1773       1783       1793CAGUUUGGCC GGGAUAUCGA CGAUACCACA GACGCCGUUA GAGAUCCCCA GACACUGGAA  Q  F  G   R  D  I   D  D  T  T   D  A  V   R  D  P   Q  T  L  E                          S protein mut3      1803       1813       1823       1833       1843       1853AUCCUGGACA UCACCCCUUG CAGCUUCGGC GGAGUGUCUG UGAUCACCCC UGGCACCAAC  I  L  D   I  T  P   C  S  F  G   G  V  S   V  I  T   P  G  T  N                          S protein mut3      1863       1873       1883       1893       1903       1913ACCAGCAAUC AGGUGGCAGU GCUGUACCAG GGCGUGAACU GUACCGAAGU GCCCGUGGCC  T  S  N   Q  V  A   V  L  Y  Q   G  V  N   C  T  E   V  P  V  A                          S protein mut3      1923       1933       1943       1953       1963       1973AUUCACGCCG AUCAGCUGAC ACCUACAUGG CGGGUGUACU CCACCGGCAG CAAUGUGUUU  I  H  A   D  Q  L   T  P  T  W   R  V  Y   S  T  G   S  N  V  F                          S protein mut3      1983       1993       2003       2013       2023       2033CAGACCAGAG CCGGCUGUCU GAUCGGAGCC GAGCACGUGA ACAAUAGCUA CGAGUGCGAC  Q  T  R   A  G  C   L  I  G  A   E  H  V   N  N  S   Y  E  C  D                          S protein mut3      2043       2053       2063       2073       2083       2093AUCCCCAUCG GCGCUGGAAU CUGCGCCAGC UACCAGACAC AGACAAACAG CCACCGGAGA  I  P  I   G  A  G   I  C  A  S   Y  Q  T   Q  T  N   S  H  R  R                          S protein mut3      2103       2113       2123       2133       2143       2153GCCAGAAGCG UGGCCAGCCA GAGCAUCAUU GCCUACACAA UGUCUCUGGG CGCCGAGAAC  A  R  S   V  A  S   Q  S  I  I   A  Y  T   M  S  L   G  A  E  N                          S protein mut3      2163       2173       2183       2193       2203       2213AGCGUGGCCU ACUCCAACAA CUCUAUCGCU AUCCCCAUCA ACUUCACCAU CAGCGUGACC  S  V  A   Y  S  N   N  S  I  A   I  P  I   N  F  T   I  S  V  T                          S protein mut3      2223       2233       2243       2253       2263       2273ACAGAGAUCC UGCCUGUGUC CAUGACCAAG ACCAGCGUGG ACUGCACCAU GUACAUCUGC  T  E  I   L  P  V   S  M  T  K   T  S  V   D  C  T   M  Y  I  C                          S protein mut3      2283       2293       2303       2313       2323       2333GGCGAUUCCA CCGAGUGCUC CAACCUGCUG CUGCAGUACG GCAGCUUCUG CACCCAGCUG  G  D  S   T  E  C   S  N  L  L   L  Q  Y   G  S  F   C  T  Q  L                          S protein mut3      2343       2353       2363       2373       2383       2393AAUAGAGCCC UGACAGGGAU CGCCGUGGAA CAGGACAAGA ACACCCAAGA GGUGUUCGCC  N  R  A   L  T  G   I  A  V  E   Q  D  K   N  T  Q   E  V  F  A                          S protein mut3      2403       2413       2423       2433       2443       2453CAAGUGAAGC AGAUCUACAA GACCCCUCCU AUCAAGGACU UCGGCGGCUU CAAUUUCAGC  Q  V  K   Q  I  Y   K  T  P  P   I  K  D   F  G  G   F  N  F  S                          S protein mut3      2463       2473       2483       2493       2503       2513CAGAUUCUGC CCGAUCCUAG CAAGCCCAGC AAGCGGAGCU UCAUCGAGGA CCUGCUGUUC  Q  I  L   P  D  P   S  K  P  S   K  R  S   F  I  E   D  L  L  F                          S protein mut3      2523       2533       2543       2553       2563       2573AACAAAGUGA CACUGGCCGA CGCCGGCUUC AUCAAGCAGU AUGGCGAUUG UCUGGGCGAC  N  K  V   T  L  A   D  A  G  F   I  K  Q   Y  G  D   C  L  G  D                          S protein mut3      2583       2593       2603       2613       2623       2633AUUGCCGCCA GGGAUCUGAU UUGCGCCCAG AAGUUUAACG GACUGACAGU GCUGCCUCCU  I  A  A   R  D  L   I  C  A  Q   K  F  N   G  L  T   V  L  P  P                          S protein mut3      2643       2653       2663       2673       2683       2693CUGCUGACCG AUGAGAUGAU CGCCCAGUAC ACAUCUGCCC UGCUGGCCGG CACAAUCACA  L  L  T   D  E  M   I  A  Q  Y   T  S  A   L  L  A   G  T  I  T                          S protein mut3      2703       2713       2723       2733       2743       2753AGCGGCUGGA CAUUUGGAGC AGGCGCCGCU CUGCAGAUCC CCUUUGCUAU GCAGAUGGCC  S  G  W   T  F  G   A  G  A  A   L  Q  I   P  F  A   M  Q  M  A                          S protein mut3      2763       2773       2783       2793       2803       2813UACCGGUUCA ACGGCAUCGG AGUGACCCAG AAUGUGCUGU ACGAGAACCA GAAGCUGAUC  Y  R  F   N  G  I   G  V  T  Q   N  V  L   Y  E  N   Q  K  L  I                          S protein mut3      2823       2833       2843       2853       2863       2873GCCAACCAGU UCAACAGCGC CAUCGGCAAG AUCCAGGACA GCCUGAGCAG CACAGCAAGC  A  N  Q   F  N  S   A  I  G  K   I  Q  D   S  L  S   S  T  A  S                          S protein mut3      2883       2893       2903       2913       2923       2933GCCCUGGGAA AGCUGCAGGA CGUGGUCAAC CAGAAUGCCC AGGCACUGAA CACCCUGGUC  A  L  G   K  L  Q   D  V  V  N   Q  N  A   Q  A  L   N  T  L  V                          S protein mut3      2943       2953       2963       2973       2983       2993AAGCAGCUGU CCUCCAACUU CGGCGCCAUC AGCUCUGUGC UGAACGAUAU CCUGGCCAGA  K  Q  L   S  S  N   F  G  A  I   S  S  V   L  N  D   I  L  A  R                          S protein mut3      3003       3013       3023       3033       3043       3053CUGGACCCUC CUGAGGCCGA GGUGCAGAUC GACAGACUGA UCACAGGCAG ACUGCAGAGC  L  D  P   P  E  A   E  V  Q  I   D  R  L   I  T  G   R  L  Q  S                          S protein mut3      3063       3073       3083       3093       3103       3113CUCCAGACAU ACGUGACCCA GCAGCUGAUC AGAGCCGCCG AGAUUAGAGC CUCUGCCAAU  L  Q  T   Y  V  T   Q  Q  L  I   R  A  A   E  I  R   A  S  A  N                          S protein mut3      3123       3133       3143       3153       3163       3173CUGGCCGCCA CCAAGAUGUC UGAGUGUGUG CUGGGCCAGA GCAAGAGAGU GGACUUUUGC  L  A  A   T  K  M   S  E  C  V   L  G  Q   S  K  R   V  D  F  C                          S protein mut3      3183       3193       3203       3213       3223       3233GGCAAGGGCU ACCACCUGAU GAGCUUCCCU CAGUCUGCCC CUCACGGCGU GGUGUUUCUG  G  K  G   Y  H  L   M  S  F  P   Q  S  A   P  H  G   V  V  F  L                          S protein mut3      3243       3253       3263       3273       3283       3293CACGUGACAU AUGUGCCCGC UCAAGAGAAG AAUUUCACCA CCGCUCCAGC CAUCUGCCAC  H  V  T   Y  V  P   A  Q  E  K   N  F  T   T  A  P   A  I  C  H                          S protein mut3      3303       3313       3323       3333       3343       3353GACGGCAAAG CCCACUUUCC UAGAGAAGGC GUGUUCGUGU CCAACGGCAC CCAUUGGUUC  D  G  K   A  H  F   P  R  E  G   V  F  V   S  N  G   T  H  W  F                          S protein mut3      3363       3373       3383       3393       3403       3413GUGACACAGC GGAACUUCUA CGAGCCCCAG AUCAUCACCA CCCACAACAC CUUCGUGUCU  V  T  Q   R  N  F   Y  E  P  Q   I  I  T   T  H  N   T  F  V  S                          S protein mut3      3423       3433       3443       3453       3463       3473GGCAACUGCG ACGUCGUGAU CGGCAUUGUG AACAAUACCG UGUACGACCC UCUGCAGCCC  G  N  C   D  V  V   I  G  I  V   N  N  T   V  Y  D   P  L  Q  P                          S protein mut3      3483       3493       3503       3513       3523       3533GAGCUGGACA GCUUCAAAGA GGAACUGGAC AAGUACUUUA AGAACCACAC AAGCCCCGAC  E  L  D   S  F  K   E  E  L  D   K  Y  F   K  N  H   T  S  P  D                          S protein mut3      3543       3553       3563       3573       3583       3593GUGGACCUGG GCGAUAUCAG CGGAAUCAAU GCCAGCGUCG UGAACAUCCA GAAAGAGAUC  V  D  L   G  D  I   S  G  I  N   A  S  V   V  N  I   Q  K  E  I                          S protein mut3      3603       3613       3623       3633       3643       3653GACCGGCUGA ACGAGGUGGC CAAGAAUCUG AACGAGAGCC UGAUCGACCU GCAAGAACUG  D  R  L   N  E  V   A  K  N  L   N  E  S   L  I  D   L  Q  E  L                          S protein mut3      3663       3673       3683       3693       3703       3713GGGAAGUACG AGCAGUACAU CAAGUGGCCC UGGUACAUCU GGCUGGGCUU UAUCGCCGGA  G  K  Y   E  Q  Y   I  K  W  P   W  Y  I   W  L  G   F  I  A  G                          S protein mut3      3723       3733       3743       3753       3763       3773CUGAUUGCCA UCGUGAUGGU CACAAUCAUG CUGUGUUGCA UGACCAGCUG CUGUAGCUGC  L  I  A   I  V  M   V  T  I  M   L  C  C   M  T  S   C  C  S  C                          S protein mut3      3783       3793       3803       3813       3823       3833CUGAAGGGCU GUUGUAGCUG UGGCAGCUGC UGCAAGUUCG ACGAGGACGA UUCUGAGCCC  L  K  G   C  C  S   C  G  S  C   C  K  F   D  E  D   D  S  E  P                          S protein mut3      3843       3853       3863    3869GUGCUGAAGG GCGUGAAACU GCACUACACA UGAUGA  V  L  K   G  V  K   L  H  Y  T   *  *                          S protein mut3      3879       3889       3899       3909       3919       3929GAUCUGCUGG UACUGCAUGC ACGCAAUGCU AGCUGCCCCU UUCCCGUCCU GGGUACCCCG                          FI element      3939       3949       3959       3969       3979       3989AGUCUCCCCC GACCUCGGGU CCCAGGUAUG CUCCCACCUC CACCUGCCCC ACUCACCACC                          FI element      3999       4009       4019       4029       4039       4049UCUGCUAGUU CCAGACACCU CCCAAGCACG CAGCAAUGCA GCUCAAAACG CUUAGCCUAG                          FI element      4059       4069       4079       4089       4099       4109CCACACCCCC ACGGGAAACA GCAGUGAUUA ACCUUUAGCA AUAAACGAAA GUUUAACUAA                          FI element      4119       4129       4139       4149       4159  4164GCUAUACUAA CCCCAGGGUU GGUCAAUUUC GUGCCAGCCA CACCCUGGAG CUAGC                          FI element      4174       4184       4194       4204       4214       4224AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA GCAUAUGACU AAAAAAAAAA AAAAAAAAAA                            Poly(A)      4234       4244       4254       4264       4274AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA                            Poly(A)

Sequences of RBP020.14 are also shown in Table 4.

TABLE 4 Sequences of RBP020.14 (Alpha-specific RNA vaccine) SEQ ID NO.Brief Description Sequence  58 Amino acid sequenceMFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFof RNA-encodedHAISGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCESARS-Cov-2 S proteinFQFCNDPFLGVYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDfrom an Alpha variantGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAA(with PRO mutationsAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRat positionsFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTcorresponding toNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKK986P and V987P ofSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATSEQ ID NO: 1; i.e.,VCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIDDTTDAVRDPQTLEILDITPRO mutations atPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGApositions 983 andEHVNNSYECDIPIGAGICASYQTQTNSHRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPINF984 of SEQ ID NO: 58)TISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILARLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTHNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT**  59 RNA sequence encoding AUGUUCGUGU UCCUGGUGCU GCUGCCUCUG GUGUCCAGCC AGUGUGUGAA CCUGACCACCa SARS-CoV-2 S proteinAGAACACAGC UGCCUCCAGC CUACACCAAC AGCUUUACCA GAGGCGUGUA CUACCCCGACfrom a Alpha variantAAGGUGUUCA GAUCCAGCGU GCUGCACUCU ACCCAGGACC UGUUCCUGCC UUUCUUCAGCAACGUGACCU GGUUCCACGC CAUCUCCGGC ACCAAUGGCA CCAAGAGAUU CGACAACCCCGUGCUGCCCU UCAACGACGG GGUGUACUUU GCCAGCACCG AGAAGUCCAA CAUCAUCAGAGGCUGGAUCU UCGGCACCAC ACUGGACAGC AAGACCCAGA GCCUGCUGAU CGUGAACAACGCCACCAACG UGGUCAUCAA AGUGUGCGAG UUCCAGUUCU GCAACGACCC CUUCCUGGGCGUCUACCACA AGAACAACAA GAGCUGGAUG GAAAGCGAGU UCCGGGUGUA CAGCAGCGCCAACAACUGCA CCUUCGAGUA CGUGUCCCAG CCUUUCCUGA UGGACCUGGA AGGCAAGCAGGGCAACUUCA AGAACCUGCG CGAGUUCGUG UUUAAGAACA UCGACGGCUA CUUCAAGAUCUACAGCAAGC ACACCCCUAU CAACCUCGUG CGGGAUCUGC CUCAGGGCUU CUCUGCUCUGGAACCCCUGG UGGAUCUGCC CAUCGGCAUC AACAUCACCC GGUUUCAGAC ACUGCUGGCCCUGCACAGAA GCUACCUGAC ACCUGGCGAU AGCAGCAGCG GAUGGACAGC UGGUGCCGCCGCUUACUAUG UGGGCUACCU GCAGCCUAGA ACCUUCCUGC UGAAGUACAA CGAGAACGGCACCAUCACCG ACGCCGUGGA UUGUGCUCUG GAUCCUCUGA GCGAGACAAA GUGCACCCUGAAGUCCUUCA CCGUGGAAAA GGGCAUCUAC CAGACCAGCA ACUUCCGGGU GCAGCCCACCGAAUCCAUCG UGCGGUUCCC CAAUAUCACC AAUCUGUGCC CCUUCGGCGA GGUGUUCAAUGCCACCAGAU UCGCCUCUGU GUACGCCUGG AACCGGAAGC GGAUCAGCAA UUGCGUGGCCGACUACUCCG UGCUGUACAA CUCCGCCAGC UUCAGCACCU UCAAGUGCUA CGGCGUGUCCCCUACCAAGC UGAACGACCU GUGCUUCACA AACGUGUACG CCGACAGCUU CGUGAUCCGGGGAGAUGAAG UGCGGCAGAU UGCCCCUGGA CAGACAGGCA AGAUCGCCGA CUACAACUAC AAGCUGCCCG ACGACUUCAC CGGCUGUGUG AUUGCCUGGA ACAGCAACAA CCUGGACUCCAAAGUCGGCG GCAACUACAA UUACCUGUAC CGGCUGUUCC GGAAGUCCAA UCUGAAGCCCUUCGAGCGGG ACAUCUCCAC CGAGAUCUAU CAGGCCGGCA GCACCCCUUG UAACGGCGUG GAAGGCUUCA ACUGCUACUU CCCACUGCAG UCCUACGGCU UUCAGCCCAC AUACGGCGUGGGCUAUCAGC CCUACAGAGU GGUGGUGCUG AGCUUCGAAC UGCUGCAUGC CCCUGCCACAGUGUGCGGCC CUAAGAAAAG CACCAAUCUC GUGAAGAACA AAUGCGUGAA CUUCAACUUCAACGGCCUGA CCGGCACCGG CGUGCUGACA GAGAGCAACA AGAAGUUCCU GCCAUUCCAGCAGUUUGGCC GGGAUAUCGA CGAUACCACA GACGCCGUUA GAGAUCCCCA GACACUGGAAAUCCUGGACA UCACCCCUUG CAGCUUCGGC GGAGUGUCUG UGAUCACCCC UGGCACCAACACCAGCAAUC AGGUGGCAGU GCUGUACCAG GGCGUGAACU GUACCGAAGU GCCCGUGGCCAUUCACGCCG AUCAGCUGAC ACCUACAUGG CGGGUGUACU CCACCGGCAG CAAUGUGUUUCAGACCAGAG CCGGCUGUCU GAUCGGAGCC CUGCGCCAGC ACAAUAGCUA CGAGUGCGAC AUCCCCAUCG GCGCUGGAAU GAGCAUCAUU GAGCACGUGA AGACAAACAG CCACCGGAGA GCCAGAAGCG UGGCCAGCCA UACCAGACAC GCCUACACAA UGUCUCUGGG CGCCGAGAACAGCGUGGCCU ACUCCAACAA CUCUAUCGCU AUCCCCAUCA ACUUCACCAU CAGCGUGACC ACAGAGAUCC UGCCUGUGUC CAUGACCAAG ACCAGCGUGG ACUGCACCAU GUACAUCUGC GGCGAUUCCA CCGAGUGCUC CAACCUGCUG CUGCAGUACG GCAGCUUCUG CACCCAGCUG AAUAGAGCCC UGACAGGGAU CGCCGUGGAA CAGGACAAGA ACACCCAAGA GGUGUUCGCC CAAGUGAAGC AGAUCUACAA GACCCCUCCU AUCAAGGACU UCGGCGGCUU CAAUUUCAGC CAGAUUCUGC CCGAUCCUAG CAAGCCCAGC AAGCGGAGCU UCAUCGAGGA CCUGCUGUUC AACAAAGUGA CACUGGCCGA CGCCGGCUUC AUCAAGCAGU AUGGCGAUUG UCUGGGCGAC AUUGCCGCCA GGGAUCUGAU UUGCGCCCAG AAGUUUAACG GACUGACAGU GCUGCCUCCU CUGCUGACCG AUGAGAUGAU CGCCCAGUAC ACAUCUGCCC UGCUGGCCGG CACAAUCACAAGCGGCUGGA CAUUUGGAGC AGGCGCCGCU CUGCAGAUCC CCUUUGCUAU GCAGAUGGCCUACCGGUUCA ACGGCAUCGG AGUGACCCAG AAUGUGCUGU ACGAGAACCA GAAGCUGAUCGCCAACCAGU UCAACAGCGC CAUCGGCAAG AUCCAGGACA GCCUGAGCAG CACAGCAAGCGCCCUGGGAA AGCUGCAGGA CGUGGUCAAC CAGAAUGCCC AGGCACUGAA CACCCUGGUCAAGCAGCUGU CCUCCAACUU CGGCGCCAUC AGCUCUGUGC UGAACGAUAU CCUGGCCAGACUGGACCCUC CUGAGGCCGA GGUGCAGAUC GACAGACUGA UCACAGGCAG ACUGCAGAGCCUCCAGACAU ACGUGACCCA GCAGCUGAUC AGAGCCGCCG AGAUUAGAGC CUCUGCCAAUCUGGCCGCCA CCAAGAUGUC UGAGUGUGUG CUGGGCCAGA GCAAGAGAGU GGACUUUUGCGGCAAGGGCU ACCACCUGAU GAGCUUCCCU CAGUCUGCCC CUCACGGCGU GGUGUUUCUGCACGUGACAU AUGUGCCCGC UCAAGAGAAG AAUUUCACCA CCGCUCCAGC CAUCUGCCACGACGGCAAAG CCCACUUUCC UAGAGAAGGC GUGUUCGUGU CCAACGGCAC CCAUUGGUUCGUGACACAGC GGAACUUCUA CGAGCCCCAG AUCAUCACCA CCCACAACAC CUUCGUGUCUGGCAACUGCG ACGUCGUGAU CGGCAUUGUG AACAAUACCG UGUACGACCC UCUGCAGCCC GAGCUGGACA GCUUCAAAGA GGAACUGGAC AAGUACUUUA AGAACCACAC AAGCCCCGAC GUGGACCUGG GCGAUAUCAG CGGAAUCAAU GCCAGCGUCG UGAACAUCCA GAAAGAGAUC GACCGGCUGA ACGAGGUGGC CAAGAAUCUG AACGAGAGCC UGAUCGACCU GCAAGAACUG GGGAAGUACG AGCAGUACAU CAAGUGGCCC UGGUACAUCU GGCUGGGCUU UAUCGCCGGA CUGAUUGCCA UCGUGAUGGU CACAAUCAUG CUGUGUUGCA UGACCAGCUG CUGUAGCUGC CUGAAGGGCU GUUGUAGCUG UGGCAGCUGC UGCAAGUUCG ACGAGGACGA UUCUGAGCCCGUGCUGAAGG GCGUGAAACU GCACUACACA UGAUGA  154 Sequence encodingATGTTCGTGT TCCTGGTGCT GCTGCCTCTG GTGTCCAGCC AGTGTGTGAA CCTGACCACC a SARS-COV-2 S proteinAGAACACAGC TGCCTCCAGC CTACACCAAC AGCTTTACCA GAGGCGTGTA CTACCCCGAC from a Alpha variantAAGGTGTTCA GATCCAGCGT GCTGCACTCT ACCCAGGACC TGTTCCTGCC TTTCTTCAGC AACGTGACCT GGTTCCACGC CATCTCCGGC ACCAATGGCA CCAAGAGATT CGACAACCCC GTGCTGCCCT TCAACGACGG GGTGTACTTT GCCAGCACCG AGAAGTCCAA CATCATCAGA GGCTGGATCT TCGGCACCAC ACTGGACAGC AAGACCCAGA GCCTGCTGAT CGTGAACAAC GCCACCAACG TGGTCATCAA AGTGTGCGAG TTCCAGTTCT GCAACGACCC CTTCCTGGGC GTCTACCACA AGAACAACAA GAGCTGGATG GAAAGCGAGT TCCGGGTGTA CAGCAGCGCC AACAACTGCA CCTTCGAGTA CGTGTCCCAG CCTTTCCTGA TGGACCTGGA AGGCAAGCAGGGCAACTTCA AGAACCTGCG CGAGTTCGTG TTTAAGAACA TCGACGGCTA CTTCAAGATCTACAGCAAGC ACACCCCTAT CAACCTCGTG CGGGATCTGC CTCAGGGCTT CTCTGCTCTGGAACCCCTGG TGGATCTGCC CATCGGCATC AACATCACCC GGTTTCAGAC ACTGCTGGCC CTGCACAGAA GCTACCTGAC ACCTGGCGAT AGCAGCAGCG GATGGACAGC TGGTGCCGCC GCTTACTATG TGGGCTACCT GCAGCCTAGA ACCTTCCTGC TGAAGTACAA CGAGAACGGC ACCATCACCG ACGCCGTGGA TTGTGCTCTG GATCCTCTGA GCGAGACAAA GTGCACCCTG AAGTCCTTCA CCGTGGAAAA GGGCATCTAC CAGACCAGCA ACTTCCGGGT GCAGCCCACC GAATCCATCG TGCGGTTCCC CAATATCACC AATCTGTGCC CCTTCGGCGA GGTGTTCAATGCCACCAGAT TCGCCTCTGT GTACGCCTGG AACCGGAAGC GGATCAGCAA TTGCGTGGCC GACTACTCCG TGCTGTACAA CTCCGCCAGC TTCAGCACCT TCAAGTGCTA CGGCGTGTCC CCTACCAAGC TGAACGACCT GTGCTTCACA AACGTGTACG CCGACAGCTT CGTGATCCGGGGAGATGAAG TGCGGCAGAT TGCCCCTGGA CAGACAGGCA AGATCGCCGA CTACAACTAC AAGCTGCCCG ACGACTTCAC CGGCTGTGTG ATTGCCTGGA ACAGCAACAA CCTGGACTCC AAAGTCGGCG GCAACTACAA TTACCTGTAC CGGCTGTTCC GGAAGTCCAA TCTGAAGCCC TTCGAGCGGG ACATCTCCAC CGAGATCTAT CAGGCCGGCA GCACCCCTTG TAACGGCGTG GAAGGCTTCA ACTGCTACTT CCCACTGCAG TCCTACGGCT TTCAGCCCAC ATACGGCGTG GGCTATCAGC CCTACAGAGT GGTGGTGCTG AGCTTCGAAC TGCTGCATGC CCCTGCCACA GTGTGCGGCC CTAAGAAAAG CACCAATCTC GTGAAGAACA AATGCGTGAA CTTCAACTTC AACGGCCTGA CCGGCACCGG CGTGCTGACA GAGAGCAACA AGAAGTTCCT GCCATTCCAG CAGTTTGGCC GGGATATCGA CGATACCACA GACGCCGTTA GAGATCCCCA GACACTGGAA ATCCTGGACA TCACCCCTTG CAGCTTCGGC GGAGTGTCTG TGATCACCCC TGGCACCAAC ACCAGCAATC AGGTGGCAGT GCTGTACCAG GGCGTGAACT GTACCGAAGT GCCCGTGGCC ATTCACGCCG ATCAGCTGAC ACCTACATGG CGGGTGTACT CCACCGGCAG CAATGTGTTTCAGACCAGAG CCGGCTGTCT GATCGGAGCC GAGCACGTGA ACAATAGCTA CGAGTGCGAC ATCCCCATCG GCGCTGGAAT CTGCGCCAGC TACCAGACAC AGACAAACAG CCACCGGAGAGCCAGAAGCG TGGCCAGCCA GAGCATCATT GCCTACACAA TGTCTCTGGG CGCCGAGAAC AGCGTGGCCT ACTCCAACAA CTCTATCGCT ATCCCCATCA ACTTCACCAT CAGCGTGACC ACAGAGATCC TGCCTGTGTC CATGACCAAG ACCAGCGTGG ACTGCACCAT GTACATCTGC GGCGATTCCA CCGAGTGCTC CAACCTGCTG CTGCAGTACG GCAGCTTCTG CACCCAGCTG AATAGAGCCC TGACAGGGAT CGCCGTGGAA CAGGACAAGA ACACCCAAGA GGTGTTCGCC CAAGTGAAGC AGATCTACAA GACCCCTCCT ATCAAGGACT TCGGCGGCTT CAATTTCAGC CAGATTCTGC CCGATCCTAG CAAGCCCAGC AAGCGGAGCT TCATCGAGGA CCTGCTGTTC AACAAAGTGA CACTGGCCGA CGCCGGCTTC ATCAAGCAGT ATGGCGATTG TCTGGGCGAC ATTGCCGCCA GGGATCTGAT TTGCGCCCAG AAGTTTAACG GACTGACAGT GCTGCCTCCT CTGCTGACCG ATGAGATGAT CGCCCAGTAC ACATCTGCCC TGCTGGCCGG CACAATCACAAGCGGCTGGA CATTTGGAGC AGGCGCCGCT CTGCAGATCC CCTTTGCTAT GCAGATGGCC TACCGGTTCA ACGGCATCGG AGTGACCCAG AATGTGCTGT ACGAGAACCA GAAGCTGATCGCCAACCAGT TCAACAGCGC CATCGGCAAG ATCCAGGACA GCCTGAGCAG CACAGCAAGCGCCCTGGGAA AGCTGCAGGA CGTGGTCAAC CAGAATGCCC AGGCACTGAA CACCCTGGTCAAGCAGCTGT CCTCCAACTT CGGCGCCATC AGCTCTGTGC TGAACGATAT CCTGGCCAGA CTGGACCCTC CTGAGGCCGA GGTGCAGATC GACAGACTGA TCACAGGCAG ACTGCAGAGC CTCCAGACAT ACGTGACCCA GCAGCTGATC AGAGCCGCCG AGATTAGAGC CTCTGCCAAT CTGGCCGCCA CCAAGATGTC TGAGTGTGTG CTGGGCCAGA GCAAGAGAGT GGACTTTTGC GGCAAGGGCT ACCACCTGAT GAGCTTCCCT CAGTCTGCCC CTCACGGCGT GGTGTTTCTG CACGTGACAT ATGTGCCCGC TCAAGAGAAG AATTTCACCA CCGCTCCAGC CATCTGCCAC GACGGCAAAG CCCACTTTCC TAGAGAAGGC GTGTTCGTGT CCAACGGCAC CCATTGGTTCGTGACACAGC GGAACTTCTA CGAGCCCCAG ATCATCACCA CCCACAACAC CTTCGTGTCTGGCAACTGCG ACGTCGTGAT CGGCATTGTG AACAATACCG TGTACGACCC TCTGCAGCCC GAGCTGGACA GCTTCAAAGA GGAACTGGAC AAGTACTTTA AGAACCACAC AAGCCCCGAC GTGGACCTGG GCGATATCAG CGGAATCAAT GCCAGCGTCG TGAACATCCA GAAAGAGATC GACCGGCTGA ACGAGGTGGC CAAGAATCTG AACGAGAGCC TGATCGACCT GCAAGAACTG GGGAAGTACG AGCAGTACAT CAAGTGGCCC TGGTACATCT GGCTGGGCTT TATCGCCGGA CTGATTGCCA TCGTGATGGT CACAATCATG CTGTGTTGCA TGACCAGCTG CTGTAGCTGC CTGAAGGGCT GTTGTAGCTG TGGCAGCTGC TGCAAGTTCG ACGAGGACGA TTCTGAGCCCGTGCTGAAGG GCGTGAAACT GCACTACACA TGATGA   60 Full length AGAAUAAACU AGUAUUCUUC UGGUCCCCAC AGACUCAGAG AGAACCCGCC ACC  sequence ofAUGUUCGUGU UCCUGGUGCU GCUGCCUCUG GUGUCCAGCC AGUGUGUGAA CCUGACCACC RBP020.14 (RNA)AGAACACAGC UGCCUCCAGC CUACACCAAC AGCUUUACCA GAGGCGUGUA CUACCCCGAC AAGGUGUUCA GAUCCAGCGU GCUGCACUCU ACCCAGGACC UGUUCCUGCC UUUCUUCAGC AACGUGACCU GGUUCCACGC CAUCUCCGGC ACCAAUGGCA CCAAGAGAUU CGACAACCCC GUGCUGCCCU UCAACGACGG GGUGUACUUU GCCAGCACCG AGAAGUCCAA CAUCAUCAGA GGCUGGAUCU UCGGCACCAC ACUGGACAGC AAGACCCAGA GCCUGCUGAU CGUGAACAAC GCCACCAACG UGGUCAUCAA AGUGUGCGAG UUCCAGUUCU GCAACGACCC CUUCCUGGGC GUCUACCACA AGAACAACAA GAGCUGGAUG GAAAGCGAGU UCCGGGUGUA CAGCAGCGCC AACAACUGCA CCUUCGAGUA CGUGUCCCAG CCUUUCCUGA UGGACCUGGA AGGCAAGCAGGGCAACUUCA AGAACCUGCG CGAGUUCGUG UUUAAGAACA UCGACGGCUA CUUCAAGAUCUACAGCAAGC ACACCCCUAU CAACCUCGUG CGGGAUCUGC CUCAGGGCUU CUCUGCUCUGGAACCCCUGG UGGAUCUGCC CAUCGGCAUC AACAUCACCC GGUUUCAGAC ACUGCUGGCCCUGCACAGAA GCUACCUGAC ACCUGGCGAU AGCAGCAGCG GAUGGACAGC UGGUGCCGCCGCUUACUAUG UGGGCUACCU GCAGCCUAGA ACCUUCCUGC UGAAGUACAA CGAGAACGGC ACCAUCACCG ACGCCGUGGA UUGUGCUCUG GAUCCUCUGA GCGAGACAAA GUGCACCCUG AAGUCCUUCA CCGUGGAAAA GGGCAUCUAC CAGACCAGCA ACUUCCGGGU GCAGCCCACC GAAUCCAUCG UGCGGUUCCC CAAUAUCACC AAUCUGUGCC CCUUCGGCGA GGUGUUCAAU GCCACCAGAU UCGCCUCUGU GUACGCCUGG AACCGGAAGC GGAUCAGCAA UUGCGUGGCC GACUACUCCG UGCUGUACAA CUCCGCCAGC UUCAGCACCU UCAAGUGCUA CGGCGUGUCC CCUACCAAGC UGAACGACCU GUGCUUCACA AACGUGUACG CCGACAGCUU CGUGAUCCGGGGAGAUGAAG UGCGGCAGAU UGCCCCUGGA CAGACAGGCA AGAUCGCCGA CUACAACUACAAGCUGCCCG ACGACUUCAC CGGCUGUGUG AUUGCCUGGA ACAGCAACAA CCUGGACUCCAAAGUCGGCG GCAACUACAA UUACCUGUAC CGGCUGUUCC GGAAGUCCAA UCUGAAGCCCUUCGAGCGGG ACAUCUCCAC CGAGAUCUAU CAGGCCGGCA GCACCCCUUG UAACGGCGUGGAAGGCUUCA ACUGCUACUU CCCACUGCAG UCCUACGGCU UUCAGCCCAC AUACGGCGUG GGCUAUCAGC CCUACAGAGU GGUGGUGCUG AGCUUCGAAC UGCUGCAUGC CCCUGCCACA GUGUGCGGCC CUAAGAAAAG CACCAAUCUC GUGAAGAACA AAUGCGUGAA CUUCAACUUCAACGGCCUGA CCGGCACCGG CGUGCUGACA GAGAGCAACA AGAAGUUCCU GCCAUUCCAG CAGUUUGGCC GGGAUAUCGA CGAUACCACA GACGCCGUUA GAGAUCCCCA GACACUGGAA AUCCUGGACA UCACCCCUUG CAGCUUCGGC GGAGUGUCUG UGAUCACCCC UGGCACCAAC ACCAGCAAUC AGGUGGCAGU GCUGUACCAG GGCGUGAACU GUACCGAAGU GCCCGUGGCC AUUCACGCCG AUCAGCUGAC ACCUACAUGG CGGGUGUACU CCACCGGCAG CAAUGUGUUUCAGACCAGAG CCGGCUGUCU GAUCGGAGCC GAGCACGUGA ACAAUAGCUA CGAGUGCGAC AUCCCCAUCG GCGCUGGAAU CUGCGCCAGC UACCAGACAC AGACAAACAG CCACCGGAGA GCCAGAAGCG UGGCCAGCCA GAGCAUCAUU GCCUACACAA UGUCUCUGGG CGCCGAGAACAGCGUGGCCU ACUCCAACAA CUCUAUCGCU AUCCCCAUCA ACUUCACCAU CAGCGUGACC ACAGAGAUCC UGCCUGUGUC CAUGACCAAG ACCAGCGUGG ACUGCACCAU GUACAUCUGC GGCGAUUCCA CCGAGUGCUC CAACCUGCUG CUGCAGUACG GCAGCUUCUG CACCCAGCUG AAUAGAGCCC UGACAGGGAU CGCCGUGGAA CAGGACAAGA ACACCCAAGA GGUGUUCGCC CAAGUGAAGC AGAUCUACAA GACCCCUCCU AUCAAGGACU UCGGCGGCUU CAAUUUCAGC CAGAUUCUGC CCGAUCCUAG CAAGCCCAGC AAGCGGAGCU UCAUCGAGGA CCUGCUGUUC AACAAAGUGA CACUGGCCGA CGCCGGCUUC AUCAAGCAGU AUGGCGAUUG UCUGGGCGAC AUUGCCGCCA GGGAUCUGAU UUGCGCCCAG AAGUUUAACG GACUGACAGU GCUGCCUCCU CUGCUGACCG AUGAGAUGAU CGCCCAGUAC ACAUCUGCCC UGCUGGCCGG CACAAUCACAAGCGGCUGGA CAUUUGGAGC AGGCGCCGCU CUGCAGAUCC CCUUUGCUAU GCAGAUGGCCUACCGGUUCA ACGGCAUCGG AGUGACCCAG AAUGUGCUGU ACGAGAACCA GAAGCUGAUCGCCAACCAGU UCAACAGCGC CAUCGGCAAG AUCCAGGACA GCCUGAGCAG CACAGCAAGCGCCCUGGGAA AGCUGCAGGA CGUGGUCAAC CAGAAUGCCC AGGCACUGAA CACCCUGGUCAAGCAGCUGU CCUCCAACUU CGGCGCCAUC AGCUCUGUGC UGAACGAUAU CCUGGCCAGACUGGACCCUC CUGAGGCCGA GGUGCAGAUC GACAGACUGA UCACAGGCAG ACUGCAGAGCCUCCAGACAU ACGUGACCCA GCAGCUGAUC AGAGCCGCCG AGAUUAGAGC CUCUGCCAAU CUGGCCGCCA CCAAGAUGUC UGAGUGUGUG CUGGGCCAGA GCAAGAGAGU GGACUUUUGC GGCAAGGGCU ACCACCUGAU GAGCUUCCCU CAGUCUGCCC CUCACGGCGU GGUGUUUCUG CACGUGACAU AUGUGCCCGC UCAAGAGAAG AAUUUCACCA CCGCUCCAGC CAUCUGCCAC GACGGCAAAG CCCACUUUCC UAGAGAAGGC GUGUUCGUGU CCAACGGCAC CCAUUGGUUC GUGACACAGC GGAACUUCUA CGAGCCCCAG AUCAUCACCA CCCACAACAC CUUCGUGUCUGGCAACUGCG ACGUCGUGAU CGGCAUUGUG AACAAUACCG UGUACGACCC UCUGCAGCCCGAGCUGGACA GCUUCAAAGA GGAACUGGAC AAGUACUUUA AGAACCACAC AAGCCCCGACGUGGACCUGG GCGAUAUCAG CGGAAUCAAU GCCAGCGUCG UGAACAUCCA GAAAGAGAUCGACCGGCUGA ACGAGGUGGC CAAGAAUCUG AACGAGAGCC UGAUCGACCU GCAAGAACUGGGGAAGUACG AGCAGUACAU CAAGUGGCCC UGGUACAUCU GGCUGGGCUU UAUCGCCGGA CUGAUUGCCA UCGUGAUGGU CACAAUCAUG CUGUGUUGCA UGACCAGCUG CUGUAGCUGC CUGAAGGGCU GUUGUAGCUG UGGCAGCUGC UGCAAGUUCG ACGAGGACGA UUCUGAGCCC GUGCUGAAGG GCGUGAAACU GCACUACACA UGAUGA GAUCUGCUGG UACUGCAUGC ACGCAAUGCU AGCUGCCCCU UUCCCGUCCU GGGUACCCCGAGUCUCCCCC GACCUCGGGU CCCAGGUAUG CUCCCACCUC CACCUGCCCC ACUCACCACC UCUGCUAGUU CCAGACACCU CCCAAGCACG CAGCAAUGCA GCUCAAAACG CUUAGCCUAG CCACACCCCC ACGGGAAACA GCAGUGAUUA ACCUUUAGCA AUAAACGAAA GUUUAACUAA GCUAUACUAA CCCCAGGGUU GGUCAAUUUC GUGCCAGCCA CACCCUGGAG CUAGC AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA GCAUAUGACU AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 155 Full length AGAATAAACT AGTATTCTTC TGGTCCCCAC AGACTCAGAG AGAACCCGCC ACC  sequence ofATGTTCGTGT TCCTGGTGCT GCTGCCTCTG GTGTCCAGCC AGTGTGTGAA CCTGACCACC RBP020.14 (DNA)AGAACACAGC TGCCTCCAGC CTACACCAAC AGCTTTACCA GAGGCGTGTA CTACCCCGAC AAGGTGTTCA GATCCAGCGT GCTGCACTCT ACCCAGGACC TGTTCCTGCC TTTCTTCAGCAACGTGACCT GGTTCCACGC CATCTCCGGC ACCAATGGCA CCAAGAGATT CGACAACCCC GTGCTGCCCT TCAACGACGG GGTGTACTTT GCCAGCACCG AGAAGTCCAA CATCATCAGA GGCTGGATCT TCGGCACCAC ACTGGACAGC AAGACCCAGA GCCTGCTGAT CGTGAACAAC GCCACCAACG TCGTCATCAA AGTGTGCGAG TTCCAGTTCT GCAACGACCC CTTCCTGGGC GTCTACCACA AGAACAACAA GAGCTGGATG GAAAGCGAGT TCCGGGTGTA CAGCAGCGCC AACAACTGCA CCTTCGAGTA CGTGTCCCAG CCTTTCCTGA TGGACCTGGA AGGCAAGCAG GGCAACTTCA AGAACCTGCG CGAGTTCGTG TTTAAGAACA TCGACGGCTA CTTCAAGATC TACAGCAAGC ACACCCCTAT CAACCTCGTG CGGGATCTGC CTCAGGGCTT CTCTGCTCTG GAACCCCTGG TGGATCTGCC CATCGGCATC AACATCACCC GGTTTCAGAC ACTGCTGGCC CTGCACAGAA GCTACCTGAC ACCTGGCGAT AGCAGCAGCG GATGGACAGC TGGTGCCGCC GCTTACTATG TGGGCTACCT GCAGCCTAGA ACCTTCCTGC TGAAGTACAA CGAGAACGGC ACCATCACCG ACGCCGTGGA TTGTGCTCTG GATCCTCTGA GCGAGACAAA GTGCACCCTG AAGTCCTTCA CCGTGGAAAA GGGCATCTAC CAGACCAGCA ACTTCCGGGT GCAGCCCACC GAATCCATCG TGCGGTTCCC CAATATCACC AATCTGTGCC CCTTCGGCGA GGTGTTCAAT GCCACCAGAT TCGCCTCTGT GTACGCCTGG AACCGGAAGC GGATCAGCAA TTGCGTGGCC GACTACTCCG TGCTGTACAA CTCCGCCAGC TTCAGCACCT TCAAGTGCTA CGGCGTGTCC CCTACCAAGC TGAACGACCT GTGCTTCACA AACGTGTACG CCGACAGCTT CGTGATCCGGGGAGATGAAG TGCGGCAGAT TGCCCCTGGA CAGACAGGCA AGATCGCCGA CTACAACTAC AAGCTGCCCG ACGACTTCAC CGGCTGTGTG ATTGCCTGGA ACAGCAACAA CCTGGACTCC AAAGTCGGCG GCAACTACAA TTACCTGTAC CGGCTGTTCC GGAAGTCCAA TCTGAAGCCC TTCGAGCGGG ACATCTCCAC CGAGATCTAT CAGGCCGGCA GCACCCCTTG TAACGGCGTG GAAGGCTTCA ACTGCTACTT CCCACTGCAG TCCTACGGCT TTCAGCCCAC ATACGGCGTG GGCTATCAGC CCTACAGAGT GGTGGTGCTG AGCTTCGAAC TGCTGCATGC CCCTGCCACA GTGTGCGGCC CTAAGAAAAG CACCAATCTC GTGAAGAACA AATGCGTGAA CTTCAACTTC AACGGCCTGA CCGGCACCGG CGTGCTGACA GAGAGCAACA AGAAGTTCCT GCCATTCCAG CAGTTTGGCC GGGATATCGA CGATACCACA GACGCCGTTA GAGATCCCCA GACACTGGAA ATCCTGGACA TCACCCCTTG CAGCTTCGGG GGAGTGTCTG TGATCACCCC TGGCACCAAC ACCAGCAATC AGGTGGCAGT GCTGTACCAG GGCGTGAACT GTACCGAAGT GCCCGTGGCCATTCACGCCG ATCAGCTGAC ACCTACATGG CGGGTGTACT CCACCGGCAG CAATGTGTTTCAGACCAGAG CCGGCTGTCT GATCGGAGCC GAGCACGTGA ACAATAGCTA CGAGTGCGACATCCCCATCG GCGCTGGAAT CTGCGCCAGC TACCAGACAC AGACAAACAG CCACCGGAGAGCCAGAAGCG TGGCCAGCCA GAGCATCATT GCCTACACAA TGTCTCTGGG CGCCGAGAACAGCGTGGCCT ACTCCAACAA CTCTATCGCT ATCCCCATCA ACTTCACCAT CAGCGTGACCACAGAGATCC TGCCTGTGTC CATGACCAAG ACCAGCGTGG ACTGCACCAT GTACATCTGCGGCGATTCCA CCGAGTGCTC CAACCTGCTG CTGCAGTACG GCAGCTTCTG CACCCAGCTGAATAGAGCCC TGACAGGGAT CGCCGTGGAA CAGGACAAGA ACACCCAAGA GGTGTTCGCCCAAGTGAAGC AGATCTACAA GACCCCTCCT ATCAAGGACT TCGGCGGCTT CAATTTCAGCCAGATTCTGC CCGATCCTAG CAAGCCCAGC AAGCGGAGCT TCATCGAGGA CCTGCTGTTCAACAAAGTGA CACTGGCCGA CGCCGGCTTC ATCAAGCAGT ATGGCGATTG TCTGGGCGACATTGCCGCCA GGGATCTGAT TTGCGCCCAG AAGTTTAACG GACTGACAGT GCTGCCTCCTCTGCTGACCG ATGAGATGAT CGCCCAGTAC ACATCTGCCC TGCTGGCCGG CACAATCACAAGCGGCTGGA CATTTGGAGC AGGCGCCGCT CTGCAGATCC CCTTTGCTAT GCAGATGGCCTACCGGTTCA ACGGCATCGG AGTGACCCAG AATGTGCTGT ACGAGAACCA GAAGCTGATCGCCAACCAGT TCAACAGCGC CATCGGCAAG ATCCAGGACA GCCTGAGCAG CACAGCAAGCGCCCTGGGAA AGCTGCAGGA CGTGGTCAAC CAGAATGCCC AGGCACTGAA CACCCTGGTCAAGCAGCTGT CCTCCAACTT CGGCGCCATC AGCTCTGTGC TGAACGATAT CCTGGCCAGACTGGACCCTC CTGAGGCCGA GGTGCAGATC GACAGACTGA TCACAGGCAG ACTGCAGAGCCTCCAGACAT ACGTGACCCA GCAGCTGATC AGAGCCGCCG AGATTAGAGC CTCTGCCAATCTGGCCGCCA CCAAGATGTC TGAGTGTGTG CTGGGCCAGA GCAAGAGAGT GGACTTTTGCGGCAAGGGCT ACCACCTGAT GAGCTTCCCT CAGTCTGCCC CTCACGGCGT GGTGTTTCTGCACGTGACAT ATGTGCCCGC TCAAGAGAAG AATTTCACCA CCGCTCCAGC CATCTGCCACGACGGCAAAG CCCACTTTCC TAGAGAAGGC GTGTTCGTGT CCAACGGCAC CCATTGGTTCGTGACACAGC GGAACTTCTA CGAGCCCCAG ATCATCACCA CCCACAACAC CTTCGTGTCTGGCAACTGCG ACGTCGTGAT CGGCATTGTG AACAATACCG TGTACGACCC TCTGCAGCCCGAGCTGGACA GCTTCAAAGA GGAACTGGAC AAGTACTTTA AGAACCACAC AAGCCCCGACGTGGACCTGG GCGATATCAG CGGAATCAAT GCCAGCGTCG TGAACATCCA GAAAGAGATCGACCGGCTGA ACGAGGTGGC CAAGAATCTG AACGAGAGCC TGATCGACCT GCAAGAACTGGGGAAGTACG AGCAGTACAT CAAGTGGCCC TGGTACATCT GGCTGGGCTT TATCGCCGGACTGATTGCCA TCGTGATGGT CACAATCATG CTGTGTTGCA TGACCAGCTG CTGTAGCTGCCTGAAGGGCT GTTGTAGCTG TGGCAGCTGC TGCAAGTTCG ACGAGGACGA TTCTGAGCCCGTGCTGAAGG GCGTGAAACT GCACTACACA TGATGAGATCTGCTGG TACTGCATGC ACGCAATGCT AGCTGCCCCT TTCCCGTCCT GGGTACCCCGAGTCTCCCCC GACCTCGGGT CCCAGGTATG CTCCCACCTC CACCTGCCCC ACTCACCACCTCTGCTAGTT CCAGACACCT CCCAAGCACG CAGCAATGCA GCTCAAAACG CTTAGCCTAGCCACACCCCC ACGGGAAACA GCAGTGATTA ACCTTTAGCA ATAAACGAAA GTTTAACTAAGCTATACTAA CCCCAGGGTT GGTCAATTTC GTGCCAGCCA CACCCTGGAG CTAGCAAAAAAAAAA AAAAAAAAAA AAAAAAAAAA GCATATGACT AAAAAAAAAA AAAAAAAAAAAAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA

Nucleotide Sequence of RBP020.16 (Delta-Specific Vaccine)

Nucleotide sequence (SEQ ID NO: 63) is shown with individual sequenceelements as indicated in bold letters. In addition, the sequence of thetranslated protein (SEQ ID NO: 61) is shown in italic letters below thecoding nucleotide sequence (*=stop codon). Red text indicates pointmutations in both the nucleotide and amino acid sequences.

        10         20         30         40         50  53AGAATAAACT AGTATTCTTC TGGTCCCCAC AGACTCAGAG AGAACCCGCC ACC                          hAg-Kozak        63         73         83         93        103        113ATGTTCGTGT TCCTGGTGCT GCTGCCTCTG GTGTCCAGCC AGTGTGTGAA CCTGAGAACC  M  F  V   F  L  V   L  L  P  L   V  S  S   Q  C  V   N  L  R  T                          S protein mut4       123        133        143        153        163        173AGAACACAGC TGCCTCCAGC CTACACCAAC AGCTTTACCA GAGGCGTGTA CTACCCCGAC  R  T  Q   L  P  P   A  Y  T  N   S  F  T   R  G  V   Y  Y  P  D                          S protein mut4       183        193        203        213        223        233AAGGTGTTCA GATCCAGCGT GCTGCACTCT ACCCAGGACC TGTTCCTGCC TTTCTTCAGC  K  V  F   R  S  S   V  L  H  S   T  Q  D   L  F  L   P  F  F  S                          S protein mut4       243        253        263        273        283        293AACGTGACCT GGTTCCACGC CATCCACGTG TCCGGCACCA ATGGCACCAA GAGATTCGAC  N  V  T   W  F  H   A  I  H  V   S  G  T   N  G  T   K  R  F  D                          S protein mut4       303        313        323        333        343        353AACCCCGTGC TGCCCTTCAA CGACGGGGTG TACTTTGCCA GCACCGAGAA GTCCAACATC  N  P  V   L  P  F   N  D  G  V   Y  F  A   S  T  E   K  S  N  I                          S protein mut4       363        373        383        393        403        413ATCAGAGGCT GGATCTTCGG CACCACACTG GACAGCAAGA CCCAGAGCCT GCTGATCGTG  I  R  G   W  I  F   G  T  T  L   D  S  K   T  Q  S   L  L  I  V                          S protein mut4       423        433        443        453        463        473AACAACGCCA CCAACGTGGT CATCAAAGTG TGCGAGTTCC AGTTCTGCAA CGACCCCTTC  N  N  A   T  N  V   V  I  K  V   C  E  F   Q  F  C   N  D  P  F                          S protein mut4       483        493        503        513        523        533CTGGACGTCT ACTACCACAA GAACAACAAG AGCTGGATGG AAAGCGGCGT GTACAGCAGC  L  D  V   Y  Y  H   K  N  N  K   S  W  M   E  S  G   V  Y  S  S                          S protein mut4       543        553        563        573        583        593GCCAACAACT GCACCTTCGA GTACGTGTCC CAGCCTTTCC TGATGGACCT GGAAGGCAAG  A  N  N   C  T  F   E  Y  V  S   Q  P  F   L  M  D   L  E  G  K                          S protein mut4       603        613        623        633        643        653CAGGGCAACT TCAAGAACCT GCGCGAGTTC GTGTTTAAGA ACATCGACGG CTACTTCAAG  Q  G  N   F  K  N   L  R  E  F   V  F  K   N  I  D   G  Y  F  K                          S protein mut4       663        673        683        693        703        713ATCTACAGCA AGCACACCCC TATCAACCTC GTGCGGGATC TGCCTCAGGG CTTCTCTGCT  I  Y  S   K  H  T   P  I  N  L   V  R  D   L  P  Q   G  F  S  A                          S protein mut4       723        733        743        753        763        773CTGGAACCCC TGGTGGATCT GCCCATCGGC ATCAACATCA CCCGGTTTCA GACACTGCTG  L  E  P   L  V  D   L  P  I  G   I  N  I   T  R  F   Q  T  L  L                          S protein mut4       783        793        803        813        823        833GCCCTGCACA GAAGCTACCT GACACCTGGC GATAGCAGCA GCGGATGGAC AGCTGGTGCC  A  L  H   R  S  Y   L  T  P  G   D  S  S   S  G  W   T  A  G  A                          S protein mut4       843        853        863        873        883        893GCCGCTTACT ATGTGGGCTA CCTGCAGCCT AGAACCTTCC TGCTGAAGTA CAACGAGAAC   A  A  Y   Y  V  G   Y  L  Q  P   R  T  F   L  L  K   Y  N  E  N                          S protein mut4       903        913        923        933        943        953GGCACCATCA CCGACGCCGT GGATTGTGCT CTGGATCCTC TGAGCGAGAC AAAGTGCACC  G  T  I   T  D  A   V  D  C  A   L  D  P   L  S  E   T  K  C  T                          S protein mut4       963        973        983        993       1003       1013CTGAAGTCCT TCACCGTGGA AAAGGGCATC TACCAGACCA GCAACTTCCG GGTGCAGCCC  L  K  S   F  T  V   E  K  G  I   Y  Q  T   S  N  F   R  V  Q  P                          S protein mut4      1023       1033       1043       1053       1063       1073ACCGAATCCA TCGTGCGGTT CCCCAATATC ACCAATCTGT GCCCCTTCGG CGAGGTGTTC  T  E  S   I  V  R   F  P  N  I   T  N  L   C  P  F   G  E  V  F                          S protein mut4      1083       1093       1103       1113       1123       1133AATGCCACCA GATTCGCCTC TGTGTACGCC TGGAACCGGA AGCGGATCAG CAATTGCGTG  N  A  T   R  F  A   S  V  Y  A   W  N  R   K  R  I   S  N  C  V                          S protein mut4      1143       1153       1163       1173       1183       1193GCCGACTACT CCGTGCTGTA CAACTCCGCC AGCTTCAGCA CCTTCAAGTG CTACGGCGTG  A  D  Y   S  V  L   Y  N  S  A   S  F  S   T  F  K   C  Y  G  V                          S protein mut4      1203       1213       1223       1233       1243       1253TCCCCTACCA AGCTGAACGA CCTGTGCTTC ACAAACGTGT ACGCCGACAG CTTCGTGATC  S  P  T   K  L  N   D  L  C  F   T  N  V   Y  A  D   S  F  V  I                          S protein mut4      1263       1273       1283       1293       1303       1313CGGGGAGATG AAGTGCGGCA GATTGCCCCT GGACAGACAG GCAAGATCGC CGACTACAAC  R  G  D   E  V  R   Q  I  A  P   G  Q  T   G  K  I   A  D  Y  N                          S protein mut4      1323       1333       1343       1353       1363       1373TACAAGCTGC CCGACGACTT CACCGGCTGT GTGATTGCCT GGAACAGCAA CAACCTGGAC  Y  K  L   P  D  D   F  T  G  C   V  I  A   W  N  S   N  N  L  D                          S protein mut4      1383       1393       1403       1413       1423       1433TCCAAAGTCG GCGGCAACTA CAATTACAGG TACCGGCTGT TCCGGAAGTC CAATCTGAAG  S  K  V   G  G  N   Y  N  Y  R   Y  R  L   F  R  K   S  N  L  K                          S protein mut4      1443       1453       1463       1473       1483       1493CCCTTCGAGC GGGACATCTC CACCGAGATC TATCAGGCCG GCAGCAAGCC TTGTAACGGC  P  F  E   R  D  I   S  T  E  I   Y  Q  A   G  S  K   P  C  N  G                          S protein mut4      1503       1513       1523       1533       1543       1553GTGGAAGGCT TCAACTGCTA CTTCCCACTG CAGTCCTACG GCTTTCAGCC CACAAATGGC  V  E  G   F  N  C   Y  F  P  L   Q  S  Y   G  F  Q   P  T  N  G                          S protein mut4      1563       1573       1583       1593       1603       1613GTGGGCTATC AGCCCTACAG AGTGGTGGTG CTGAGCTTCG AACTGCTGCA TGCCCCTGCC  V  G  Y   Q  P  Y   R  V  V  V   L  S  F   E  L  L   H  A  P  A                          S protein mut4      1623       1633       1643       1653       1663       1673ACAGTGTGCG GCCCTAAGAA AAGCACCAAT CTCGTGAAGA ACAAATGCGT GAACTTCAAC  T  V  C   G  P  K   K  S  T  N   L  V  K   N  K  C   V  N  F  N                          S protein mut4      1683       1693       1703       1713       1723       1733TTCAACGGCC TGACCGGCAC CGGCGTGCTG ACAGAGAGCA ACAAGAAGTT CCTGCCATTC  F  N  G   L  T  G   T  G  V  L   T  E  S   N  K  K   F  L  P  F                          S protein mut4      1743       1753       1763       1773       1783       1793CAGCAGTTTG GCCGGGATAT CGCCGATACC ACAGACGCCG TTAGAGATCC CCAGACACTG  Q  Q  F   G  R  D   I  A  D  T   T  D  A   V  R  D   P  Q  T  L                          S protein mut4      1803       1813       1823       1833       1843       1853GAAATCCTGG ACATCACCCC TTGCAGCTTC GGCGGAGTGT CTGTGATCAC CCCTGGCACC  E  I  L   D  I  T   P  C  S  F   G  G  V   S  V  I   T  P  G  T                          S protein mut4      1863       1873       1883       1893       1903       1913AACACCAGCA ATCAGGTGGC AGTGCTGTAC CAGGGCGTGA ACTGTACCGA AGTGCCCGTG  N  T  S   N  Q  V   A  V  L  Y   Q  G  V   N  C  T   E  V  P  V                          S protein mut4      1923       1933       1943       1953       1963       1973GCCATTCACG CCGATCAGCT GACACCTACA TGGCGGGTGT ACTCCACCGG CAGCAATGTG   A  I  H   A  D  Q   L  T  P  T   W  R  V   Y  S  T   G  S  N  V                          S protein mut4      1983       1993       2003       2013       2023       2033TTTCAGACCA GAGCCGGCTG TCTGATCGGA GCCGAGCACG TGAACAATAG CTACGAGTGC  F  Q  T   R  A  G   C  L  I  G   A  E  H   V  N  N   S  Y  E  C                          S protein mut4      2043       2053       2063       2073       2083       2093GACATCCCCA TCGGCGCTGG AATCTGCGCC AGCTACCAGA CACAGACAAA CAGCAGGCGG  D  I  P   I  G  A   G  I  C  A   S  Y  Q   T  Q  T   N  S  P  R                          S protein mut4      2103       2113       2123       2133       2143       2153AGAGCCAGAA GCGTGGCCAG CCAGAGCATC ATTGCCTACA CAATGTCTCT GGGCGCCGAG  R  A  R   S  V  A   S  Q  S  I   I  A  Y   T  M  S   L  G  A  E                          S protein mut4      2163       2173       2183       2193       2203       2213AACAGCGTGG CCTACTCCAA CAACTCTATC GCTATCCCCA CCAACTTCAC CATCAGCGTG  N  S  V   A  Y  S   N  N  S  I   A  I  P   T  N  F   T  I  S  V                          S protein mut4      2223       2233       2243       2253       2263       2273ACCACAGAGA TCCTGCCTGT GTCCATGACC AAGACCAGCG TGGACTGCAC CATGTACATC  T  T  E   I  L  P   V  S  M  T   K  T  S   V  D  C   T  M  Y  I                          S protein mut4      2283       2293       2303       2313       2323       2333TGCGGCGATT CCACCGAGTG CTCCAACCTG CTGCTGCAGT ACGGCAGCTT CTGCACCCAG  C  G  D   S  T  E   C  S  N  L   L  L  Q   Y  G  S   F  C  T  Q                          S protein mut4      2343       2353       2363       2373       2383       2393CTGAATAGAG CCCTGACAGG GATCGCCGTG GAACAGGACA AGAACACCCA AGAGGTGTTC  L  N  R   A  L  T   G  I  A  V   E  Q  D   K  N  T   Q  E  V  F                          S protein mut4      2403       2413       2423       2433       2443       2453GCCCAAGTGA AGCAGATCTA CAAGACCCCT CCTATCAAGG ACTTCGGCGG CTTCAATTTC  A  Q  V   K  Q  I   Y  K  T  P   P  I  K   D  F  G   G  F  N  F                          S protein mut4      2463       2473       2483       2493       2503       2513AGCCAGATTC TGCCCGATCC TAGCAAGCCC AGCAAGCGGA GCTTCATCGA GGACCTGCTG  S  Q  I   L  P  D   P  S  K  P   S  K  R   S  F  I   E  D  L  L                          S protein mut4      2523       2533       2543       2553       2563       2573TTCAACAAAG TGACACTGGC CGACGCCGGC TTCATCAAGC AGTATGGCGA TTGTCTGGGC  F  N  K   V  T  L   A  D  A  G   F  I  K   Q  Y  G   D  C  L  G                          S protein mut4      2583       2593       2603       2613       2623       2633GACATTGCCG CCAGGGATCT GATTTGCGCC CAGAAGTTTA ACGGACTGAC AGTGCTGCCT  D  I  A   A  R  D   L  I  C  A   Q  K  F   N  G  L   T  V  L  P                          S protein mut4      2643       2653       2663       2673       2683       2693CCTCTGCTGA CCGATGAGAT GATCGCCCAG TACACATCTG CCCTGCTGGC CGGCACAATC  P  L  L   T  D  E   M  I  A  Q   Y  T  S   A  L  L   A  G  T  I                          S protein mut4      2703       2713       2723       2733       2743       2753ACAAGCGGCT GGACATTTGG AGCAGGCGCC GCTCTGCAGA TCCCCTTTGC TATGCAGATG  T  S  G   W  T  F   G  A  G  A   A  L  Q   I  P  F   A  M  Q  M                          S protein mut4      2763       2773       2783       2793       2803       2813GCCTACCGGT TCAACGGCAT CGGAGTGACC CAGAATGTGC TGTACGAGAA CCAGAAGCTG  A  Y  R   F  N  G   I  G  V  T   Q  N  V   L  Y  E   N  Q  K  L                          S protein mut4      2823       2833       2843       2853       2863       2873ATCGCCAACC AGTTCAACAG CGCCATCGGC AAGATCCAGG ACAGCCTGAG CAGCACAGCA  I  A  N   Q  F  N   S  A  I  G   K  I  Q   D  S  L   S  S  T  A                          S protein mut4      2883       2893       2903       2913       2923       2933AGCGCCCTGG GAAAGCTGCA GAACGTGGTC AACCAGAATG CCCAGGCACT GAACACCCTG  S  A  L   G  K  L   Q  H  V  V   N  Q  N   A  Q  A   L  N  T  L                          S protein mut4      2943       2953       2963       2973       2983       2993GTCAAGCAGC TGTCCTCCAA CTTCGGCGCC ATCAGCTCTG TGCTGAACGA TATCCTGAGC  V  K  Q   L  S  S   N  F  G  A   I  S  S   V  L  N   D  I  L  S                          S protein mut4      3003       3013       3023       3033       3043       3053AGACTGGACC CTCCTGAGGC CGAGGTGCAG ATCGACAGAC TGATCACAGG CAGACTGCAG  R  L  D   P  P  E   A  E  V  Q   I  D  R   L  I  T   G  R  L  Q                          S protein mut4      3063       3073       3083       3093       3103       3113AGCCTCCAGA CATACGTGAC CCAGCAGCTG ATCAGAGCCG CCGAGATTAG AGCCTCTGCC  S  L  Q   T  Y  V   T  Q  Q  L   I  R  A   A  E  I   R  A  S  A                          S protein mut4      3123       3133       3143       3153       3163       3173AATCTGGCCG CCACCAAGAT GTCTGAGTGT GTGCTGGGCC AGAGCAAGAG AGTGGACTTT  N  L  A   A  T  K   M  S  E  C   V  L  G   Q  S  K   R  V  D  F                          S protein mut4      3183       3193       3203       3213       3223       3233TGCGGCAAGG GCTACCACCT GATGAGCTTC CCTCAGTCTG CCCCTCACGG CGTGGTGTTT  C  G  K   G  Y  H   L  M  S  F   P  Q  S   A  P  H   G  V  V  F                          S protein mut4      3243       3253       3263       3273       3283       3293CTGCACGTGA CATATGTGCC CGCTCAAGAG AAGAATTTCA CCACCGCTCC AGCCATCTGC  L  H  V   T  Y  V   P  A  Q  E   K  N  F   T  T  A   P  A  I  C                          S protein mut4      3303       3313       3323       3333       3343       3353CACGACGGCA AAGCCCACTT TCCTAGAGAA GGCGTGTTCG TGTCCAACGG CACCCATTGG  H  D  G   K  A  H   F  P  R  E   G  V  F   V  S  N   G  T  H  W                          S protein mut4      3363       3373       3383       3393       3403       3413TTCGTGACAC AGCGGAACTT CTACGAGCCC CAGATCATCA CCACCGACAA CACCTTCGTG  F  V  T   Q  R  N   F  Y  E  P   Q  I  I   T  T  D   N  T  F  V                          S protein mut4      3423       3433       3443       3453       3463       3473TCTGGCAACT GCGACGTCGT GATCGGCATT GTGAACAATA CCGTGTACGA CCCTCTGCAG  S  G  N   C  D  V   V  I  G  I   V  N  N   T  V  Y   D  P  L  Q                          S protein mut4      3483       3493       3503       3513       3523       3533CCCGAGCTGG ACAGCTTCAA AGAGGAACTG GACAAGTACT TTAAGAACCA CACAAGCCCC  P  E  L   D  S  F   K  E  E  L   D  K  Y   F  K  N   H  T  S  P                          S protein mut4      3543       3553       3563       3573       3583       3593GACGTGGACC TGGGCGATAT CAGCGGAATC AATGCCAGCG TCGTGAACAT CCAGAAAGAG  D  V  D   L  G  D   I  S  G  I   N  A  S   V  V  N   I  Q  K  E                          S protein mut4      3603       3613       3623       3633       3643       3653ATCGACCGGC TGAACGAGGT GGCCAAGAAT CTGAACGAGA GCCTGATCGA CCTGCAAGAA  I  D  R   L  N  E   V  A  K  N   L  N  E   S  L  I   D  L  Q  E                          S protein mut4      3663       3673       3683       3693       3703       3713CTGGGGAAGT ACGAGCAGTA CATCAAGTGG CCCTGGTACA TCTGGCTGGG CTTTATCGCC  L  G  K   Y  E  Q   Y  I  K  W   P  W  Y   I  W  L   G  F  I  A                          S protein mut4      3723       3733       3743       3753       3763       3773GGACTGATTG CCATCGTGAT GGTCACAATC ATGCTGTGTT GCATGACCAG CTGCTGTAGC  G  L  I   A  I  V   M  V  T  I   M  L  C   C  M  T   S  C  C  S                          S protein mut4      3783       3793       3803       3813       3823       3833TGCCTGAAGG GCTGTTGTAG CTGTGGCAGC TGCTGCAAGT TCGACGAGGA CGATTCTGAG  C  L  K   G  C  C   S  C  G  S   C  C  K   F  D  E   D  D  S  E                          S protein mut4      3843       3853       3863      3872CCCGTGCTGA AGGGCGTGAA ACTGCACTAC ACATGATGA  P  V  L   K  G  V   K  L  H  Y   T  *  *                          S protein mut4      3882       3892       3902       3912       3922       3932TTTCACCTGG TACTGCATGC ACGCAATGCT AGCTGCCCCT TTCCCGTCCT GGGTACCCCG                          FI element      3942       3952       3962       3972       3982       3992AGTCTCCCCC GACCTCGGGT CCCAGGTATG CTCCCACCTC CACCTGCCCC ACTCACCACC                          FI element      4002       4012       4022       4032       4042       4052TCTGCTAGTT CCAGACACCT CCCAAGCACG CAGCAATGCA GCTCAAAACG CTTAGCCTAG                          FI element      4062       4072       4082       4092       4102       4112CCACACCCCC ACGGGAAACA GCAGTGATTA ACCTTTAGCA ATAAACGAAA GTTTAACTAA                          FI element      4122       4132       4142       4152       4162  4167GCTATACTAA CCCCAGGGTT GGTCAATTTC GTGCCAGCCA CACCCTGGAG CTAGC                          FI element      4177       4187       4197       4207       4217       4227AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA GCATATGACT AAAAAAAAAA AAAAAAAAAA                             Poly(A)      4237       4247       4257       4267       4277AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA                            Poly(A)

Sequences of RBP020.14 are also shown in Table 5.

TABLE 5 Sequences of RBP020.16 (Delta-specific RNA vaccine) SEQ BriefID NO. Description Sequence  61 Amino acid sequence of RNA-MFVFLVLLPLVSSQCVNLRTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFencoded SARS-CoV-2 S proteinHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVfrom a Delta variant (with PROCEFQFCNDPFLDVYYHKNNKSWMESGVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNImutations at positionsDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAcorresponding to K986P andAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVV987P of SEQ ID NO: 1; i.e.,RFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFPRO mutations at positions 984TNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRand 985 of SEQ ID NO: 61)KSNLKPFERDISTEIYQAGSKPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSRRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQNVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT--  62 Sequence encoding a SARS-CoV-ATGTTCGTGT TCCTGGTGCT GCTGCCTCTG GTGTCCAGCC AGTGTGTGAA CCTGAGAACC2 S protein from a DeltaAGAACACAGC TGCCTCCAGC CTACACCAAC AGCTTTACCA GAGGCGTGTA CTACCCCGACvariantAAGGTGTTCA GATCCAGCGT GCTGCACTCT ACCCAGGACC TGTTCCTGCC TTTCTTCAGCAACGTGACCT GGTTCCACGC CATCCACGTG TCCGGCACCA ATGGCACCAA GAGATTCGACAACCCCGTGC TGCCCTTCAA CGACGGGGTG TACTTTGCCA GCACCGAGAA GTCCAACATCATCAGAGGCT GGATCTTCGG CACCACACTG GACAGCAAGA CCCAGAGCCT GCTGATCGTGAACAACGCCA CCAACGTGGT CATCAAAGTG TGCGAGTTCC AGTTCTGCAA CGACCCCTTCCTGGACGTCT ACTACCACAA GAACAACAAG AGCTGGATGG AAAGCGGCGT GTACAGCAGCGCCAACAACT GCACCTTCGA GTACGTGTCC CAGCCTTTCC TGATGGACCT GGAAGGCAAGCAGGGCAACT TCAAGAACCT GCGCGAGTTC GTGTTTAAGA ACATCGACGG CTACTTCAAGATCTACAGCA AGCACACCCC TATCAACCTC GTGCGGGATC TGCCTCAGGG CTTCTCTGCTCTGGAACCCC TGGTGGATCT GCCCATCGGC ATCAACATCA CCCGGTTTCA GACACTGCTGGCCCTGCACA GAAGCTACCT GACACCTGGC GATAGCAGCA GCGGATGGAC AGCTGGTGCCGCCGCTTACT ATGTGGGCTA CCTGCAGCCT AGAACCTTCC TGCTGAAGTA CAACGAGAACGGCACCATCA CCGACGCCGT GGATTGTGCT CTGGATCCTC TGAGCGAGAC AAAGTGCACCCTGAAGTCCT TCACCGTGGA AAAGGGCATC TACCAGACCA GCAACTTCCG GGTGCAGCCCACCGAATCCA TCGTGCGGTT CCCCAATATC ACCAATCTGT GCCCCTTCGG CGAGGTGTTCAATGCCACCA GATTCGCCTC TGTGTACGCC TGGAACCGGA AGCGGATCAG CAATTGCGTGGCCGACTACT CCGTGCTGTA CAACTCCGCC AGCTTCAGCA CCTTCAAGTG CTACGGCGTGTCCCCTACCA AGCTGAACGA CCTGTGCTTC ACAAACGTGT ACGCCGACAG CTTCGTGATCCGGGGAGATG AAGTGCGGCA GATTGCCCCT GGACAGACAG GCAAGATCGC CGACTACAACTACAAGCTGC CCGACGACTT CACCGGCTGT GTGATTGCCT GGAACAGCAA CAACCTGGACTCCAAAGTCG GCGGCAACTA CAATTACAGG TACCGGCTGT TCCGGAAGTC CAATCTGAAGCCCTTCGAGC GGGACATCTC CACCGAGATC TATCAGGCCG GCAGCAAGCC TTGTAACGGCGTGGAAGGCT TCAACTGCTA CTTCCCACTG CAGTCCTACG GCTTTCAGCC CACAAATGGCGTGGGCTATC AGCCCTACAG AGTGGTGGTG CTGAGCTTCG AACTGCTGCA TGCCCCTGCCACAGTGTGCG GCCCTAAGAA AAGCACCAAT CTCGTGAAGA ACAAATGCGT GAACTTCAACTTCAACGGCC TGACCGGCAC CGGCGTGCTG ACAGAGAGCA ACAAGAAGTT CCTGCCATTCCAGCAGTTTG GCCGGGATAT CGCCGATACC ACAGACGCCG TTAGAGATCC CCAGACACTGGAAATCCTGG ACATCACCCC TTGCAGCTTC GGCGGAGTGT CTGTGATCAC CCCTGGCACCAACACCAGCA ATCAGGTGGC AGTGCTGTAC CAGGGCGTGA ACTGTACCGA AGTGCCCGTGGCCATTCACG CCGATCAGCT GACACCTACA TGGCGGGTGT ACTCCACCGG CAGCAATGTGTTTCAGACCA GAGCCGGCTG TCTGATCGGA GCCGAGCACG TGAACAATAG CTACGAGTGCGACATCCCCA TCGGCGCTGG AATCTGCGCC AGCTACCAGA CACAGACAAA CAGCAGCCGGAGAGCCAGAA GCGTGGCCAG CCAGAGCATC ATTGCCTACA CAATGTCTCT GGGCGCCGAGAACAGCGTGG CCTACTCCAA CAACTCTATC GCTATCCCCA CCAACTTCAC CATCAGCGTGACCACAGAGA TCCTGCCTGT GTCCATGACC AAGACCAGCG TGGACTGCAC CATGTACATCTGCGGCGATT CCACCGAGTG CTCCAACCTG CTGCTGCAGT ACGGCAGCTT CTGCACCCAGCTGAATAGAG CCCTGACAGG GATCGCCGTG GAACAGGACA AGAACACCCA AGAGGTGTTCGCCCAAGTGA AGCAGATCTA CAAGACCCCT CCTATCAAGG ACTTCGGCGG CTTCAATTTCAGCCAGATTC TGCCCGATCC TAGCAAGCCC AGCAAGCGGA GCTTCATCGA GGACCTGCTGTTCAACAAAG TGACACTGGC CGACGCCGGC TTCATCAAGC AGTATGGCGA TTGTCTGGGCGACATTGCCG CCAGGGATCT GATTTGCGCC CAGAAGTTTA ACGGACTGAC AGTGCTGCCTCCTCTGCTGA CCGATGAGAT GATCGCCCAG TACACATCTG CCCTGCTGGC CGGCACAATCACAAGCGGCT GGACATTTGG AGCAGGCGCC GCTCTGCAGA TCCCCTTTGC TATGCAGATGGCCTACCGGT TCAACGGCAT CGGAGTGACC CAGAATGTGC TGTACGAGAA CCAGAAGCTGATCGCCAACC AGTTCAACAG CGCCATCGGC AAGATCCAGG ACAGCCTGAG CAGCACAGCAAGCGCCCTGG GAAAGCTGCA GAACGTGGTC AACCAGAATG CCCAGGCACT GAACACCCTGGTCAAGCAGC TGTCCTCCAA CTTCGGCGCC ATCAGCTCTG TGCTGAACGA TATCCTGAGCAGACTGGACC CTCCTGAGGC CGAGGTGCAG ATCGACAGAC TGATCACAGG CAGACTGCAGAGCCTCCAGA CATACGTGAC CCAGCAGCTG ATCAGAGCCG CCGAGATTAG AGCCTCTGCCAATCTGGCCG CCACCAAGAT GTCTGAGTGT GTGCTGGGCC AGAGCAAGAG AGTGGACTTTTGCGGCAAGG GCTACCACCT GATGAGCTTC CCTCAGTCTG CCCCTCACGG CGTGGTGTTTCTGCACGTGA CATATGTGCC CGCTCAAGAG AAGAATTTCA CCACCGCTCC AGCCATCTGCCACGACGGCA AAGCCCACTT TCCTAGAGAA GGCGTGTTCG TGTCCAACGG CACCCATTGGTTCGTGACAC AGCGGAACTT CTACGAGCCC CAGATCATCA CCACCGACAA CACCTTCGTGTCTGGCAACT GCGACGTCGT GATCGGCATT GTGAACAATA CCGTGTACGA CCCTCTGCAGCCCGAGCTGG ACAGCTTCAA AGAGGAACTG GACAAGTACT TTAAGAACCA CACAAGCCCCGACGTGGACC TGGGCGATAT CAGCGGAATC AATGCCAGCG TCGTGAACAT CCAGAAAGAGATCGACCGGC TGAACGAGGT GGCCAAGAAT CTGAACGAGA GCCTGATCGA CCTGCAAGAACTGGGGAAGT ACGAGCAGTA CATCAAGTGG CCCTGGTACA TCTGGCTGGG CTTTATCGCCGGACTGATTG CCATCGTGAT GGTCACAATC ATGCTGTGTT GCATGACCAG CTGCTGTAGCTGCCTGAAGG GCTGTTGTAG CTGTGGCAGC TGCTGCAAGT TCGACGAGGA CGATTCTGAGCCCGTGCTGA AGGGCGTGAA ACTGCACTAC ACATGATGA 156 RNA sequence encoding aAUGUUCGUGU UCCUGGUGCU GCUGCCUCUG GUGUCCAGCC AGUGUGUGAA CCUGAGAACCSARS-CoV-2 S protein from aAGAACACAGC UGCCUCCAGC CUACACCAAC AGCUUUACCA GAGGCGUGUA CUACCCCGACDelta variantAAGGUGUUCA GAUCCAGCGU GCUGCACUCU ACCCAGGACC UGUUCCUGCC UUUCUUCAGCAACGUGACCU GGUUCCACGC CAUCCACGUG UCCGGCACCA AUGGCACCAA GAGAUUCGACAACCCCGUGC UGCCCUUCAA CGACGGGGUG UACUUUGCCA GCACCGAGAA GUCCAACAUCAUCAGAGGCU GGAUCUUCGG CACCACACUG GACAGCAAGA CCCAGAGCCU GCUGAUCGUGAACAACGCCA CCAACGUGGU CAUCAAAGUG UGCGAGUUCC AGUUCUGCAA CGACCCCUUCCUGGACGUCU ACUACCACAA GAACAACAAG AGCUGGAUGG AAAGCGGCGU GUACAGCAGCGCCAACAACU GCACCUUCGA GUACGUGUCC CAGCCUUUCC UGAUGGACCU GGAAGGCAAGCAGGGCAACU UCAAGAACCU GCGCGAGUUC GUGUUUAAGA ACAUCGACGG CUACUUCAAGAUCUACAGCA AGCACACCCC UAUCAACCUC GUGCGGGAUC UGCCUCAGGG CUUCUCUGCUCUGGAACCCC UGGUGGAUCU GCCCAUCGGC AUCAACAUCA CCCGGUUUCA GACACUGCUGGCCCUGCACA GAAGCUACCU GACACCUGGC GAUAGCAGCA GCGGAUGGAC AGCUGGUGCCGCCGCUUACU AUGUGGGCUA CCUGCAGCCU AGAACCUUCC UGCUGAAGUA CAACGAGAACGGCACCAUCA CCGACGCCGU GGAUUGUGCU CUGGAUCCUC UGAGCGAGAC AAAGUGCACCCUGAAGUCCU UCACCGUGGA AAAGGGCAUC UACCAGACCA GCAACUUCCG GGUGCAGCCCACCGAAUCCA UCGUGCGGUU CCCCAAUAUC ACCAAUCUGU GCCCCUUCGG CGAGGUGUUCAAUGCCACCA GAUUCGCCUC UGUGUACGCC UGGAACCGGA AGCGGAUCAG CAAUUGCGUGGCCGACUACU CCGUGCUGUA CAACUCCGCC AGCUUCAGCA CCUUCAAGUG CUACGGCGUGUCCCCUACCA AGCUGAACGA CCUGUGCUUC ACAAACGUGU ACGCCGACAG CUUCGUGAUCCGGGGAGAUG AAGUGCGGCA GAUUGCCCCU GGACAGACAG GCAAGAUCGC CGACUACAACUACAAGCUGC CCGACGACUU CACCGGCUGU GUGAUUGCCU GGAACAGCAA CAACCUGGACUCCAAAGUCG GCGGCAACUA CAAUUACAGG UACCGGCUGU UCCGGAAGUC CAAUCUGAAGCCCUUCGAGC GGGACAUCUC CACCGAGAUC UAUCAGGCCG GCAGCAAGCC UUGUAACGGCGUGGAAGGCU UCAACUGCUA CUUCCCACUG CAGUCCUACG GCUUUCAGCC CACAAAUGGCGUGGGCUAUC AGCCCUACAG AGUGGUGGUG CUGAGCUUCG AACUGCUGCA UGCCCCUGCCACAGUGUGCG GCCCUAAGAA AAGCACCAAU CUCGUGAAGA ACAAAUGCGU GAACUUCAACUUCAACGGCC UGACCGGCAC CGGCGUGCUG ACAGAGAGCA ACAAGAAGUU CCUGCCAUUCCAGCAGUUUG GCCGGGAUAU CGCCGAUACC ACAGACGCCG UUAGAGAUCC CCAGACACUGGAAAUCCUGG ACAUCACCCC UUGCAGCUUC GGCGGAGUGU CUGUGAUCAC CCCUGGCACCAACACCAGCA AUCAGGUGGC AGUGCUGUAC CAGGGCGUGA ACUGUACCGA AGUGCCCGUGGCCAUUCACG CCGAUCAGCU GACACCUACA UGGCGGGUGU ACUCCACCGG CAGCAAUGUGUUUCAGACCA GAGCCGGCUG UCUGAUCGGA GCCGAGCACG UGAACAAUAG CUACGAGUGCGACAUCCCCA UCGGCGCUGG AAUCUGCGCC AGCUACCAGA CACAGACAAA CAGCAGGCGGAGAGCCAGAA GCGUGGCCAG CCAGAGCAUC AUUGCCUACA CAAUGUCUCU GGGCGCCGAGAACAGCGUGG CCUACUCCAA CAACUCUAUC GCUAUCCCCA CCAACUUCAC CAUCAGCGUGACCACAGAGA UCCUGCCUGU GUCCAUGACC AAGACCAGCG UGGACUGCAC CAUGUACAUCUGCGGCGAUU CCACCGAGUG CUCCAACCUG CUGCUGCAGU ACGGCAGCUU CUGCACCCAGCUGAAUAGAG CCCUGACAGG GAUCGCCGUG GAACAGGACA AGAACACCCA AGAGGUGUUCGCCCAAGUGA AGCAGAUCUA CAAGACCCCU CCUAUCAAGG ACUUCGGCGG CUUCAAUUUCAGCCAGAUUC UGCCCGAUCC UAGCAAGCCC AGCAAGCGGA GCUUCAUCGA GGACCUGCUGUUCAACAAAG UGACACUGGC CGACGCCGGC UUCAUCAAGC AGUAUGGCGA UUGUCUGGGCGACAUUGCCG CCAGGGAUCU GAUUUGCGCC CAGAAGUUUA ACGGACUGAC AGUGCUGCCUCCUCUGCUGA CCGAUGAGAU GAUCGCCCAG UACACAUCUG CCCUGCUGGC CGGCACAAUCACAAGCGGCU GGACAUUUGG AGCAGGCGCC GCUCUGCAGA UCCCCUUUGC UAUGCAGAUGGCCUACCGGU UCAACGGCAU CGGAGUGACC CAGAAUGUGC UGUACGAGAA CCAGAAGCUGAUCGCCAACC AGUUCAACAG CGCCAUCGGC AAGAUCCAGG ACAGCCUGAG CAGCACAGCAAGCGCCCUGG GAAAGCUGCA GAACGUGGUC AACCAGAAUG CCCAGGCACU GAACACCCUGGUCAACCACC UCUCCUCCAA CUUCCCCCCC AUCACCUCUC UCCUCAACCA UAUCCUCACCAGACUGGACC CUCCUGAGGC CGAGGUGCAG AUCGACAGAC UGAUCACAGG CAGACUGCAGAGCCUCCAGA CAUACGUGAC CCAGCAGCUG AUCAGAGCCG CCGAGAUUAG AGCCUCUGCCAAUCUGGCCG CCACCAAGAU GUCUGAGUGU GUGCUGGGCC AGAGCAAGAG AGUGGACUUUUGCGGCAAGG GCUACCACCU GAUGAGCUUC CCUCAGUCUG CCCCUCACGG CGUGGUGUUUCUGCACGUGA CAUAUGUGCC CGCUCAAGAG AAGAAUUUCA CCACCGCUCC AGCCAUCUGCCACGACGGCA AAGCCCACUU UCCUAGAGAA GGCGUGUUCG UGUCCAACGG CACCCAUUGGUUCGUGACAC AGCGGAACUU CUACGAGCCC CAGAUCAUCA CCACCGACAA CACCUUCGUGUCUGGCAACU GCGACGUCGU GAUCGGCAUU GUGAACAAUA CCGUGUACGA CCCUCUGCAGCCCGAGCUGG ACAGCUUCAA AGAGGAACUG GACAAGUACU UUAAGAACCA CACAAGCCCCGACGUGGACC UGGGCGAUAU CAGCGGAAUC AAUGCCAGCG UCGUGAACAU CCAGAAAGAGAUCGACCGGC UGAACGAGGU GGCCAAGAAU CUGAACGAGA GCCUGAUCGA CCUGCAAGAACUGGGGAAGU ACGAGCAGUA CAUCAAGUGG CCCUGGUACA UCUGGCUGGG CUUUAUCGCCGGACUGAUUG CCAUCGUGAU GGUCACAAUC AUGCUGUGUU GCAUGACCAG CUGCUGUAGCUGCCUGAAGG GCUGUUGUAG CUGUGGCAGC UGCUGCAAGU UCGACGAGGA CGAUUCUGAGCCCGUGCUGA AGGGCGUGAA ACUGCACUAC ACAUGAUGA  63 Full length sequence ofAGAATAAACT AGTATTCTTC TGGTCCCCAC AGACTCAGAG AGAACCCGCC ACC RBP020.14ATGTTCGTGT TCCTGGTGCT GCTGCCTCTG GTGTCCAGCC AGTGTGTGAA CCTGAGAACCAGAACACAGC TGCCTCCAGC CTACACCAAC AGCTTTACCA GAGGCGTGTA CTACCCCGACAAGGTGTTCA GATCCAGCGT GCTGCACTCT ACCCAGGACC TGTTCCTGCC TTTCTTCAGCAACGTGACCT GGTTCCACGC CATCCACGTG TCCGGCACCA ATGGCACCAA GAGATTCGACAACCCCGTGC TGCCCTTCAA CGACGGGGTG TACTTTGCCA GCACCGAGAA GTCCAACATCATCAGAGGCT GGATCTTCGG CACCACACTG GACAGCAAGA CCCAGAGCCT GCTGATCGTGAACAACGCCA CCAACGTGGT CATCAAAGTG TGCGAGTTCC AGTTCTGCAA CGACCCCTTCCTGGACGTCT ACTACCACAA GAACAACAAG AGCTGGATGG AAAGCGSCGT GTACAGCAGCGCCAACAACT GCACCTTCGA GTACGTGTCC CAGCCTTTCC TGATGGACCT GGAAGGCAAGCAGGGCAACT TCAAGAACCT GCGCGAGTTC GTGTTTAAGA ACATCGACGG CTACTTCAAGATCTACAGCA AGCACACCCC TATCAACCTC GTGCGGGATC TGCCTCAGGG CTTCTCTGCTCTGGAACCCC TGGTGGATCT GCCCATCGGC ATCAACATCA CCCGGTTTCA GACACTGCTGGCCCTGCACA GAAGCTACCT GACACCTGGC GATAGCAGCA GCGGATGGAC AGCTGGTGCCGCCGCTTACT ATGTGGGCTA CCTGCAGCCT AGAACCTTCC TGCTGAAGTA CAACGAGAACGGCACCATCA CCGACGCCGT GGATTGTGCT CTGGATCCTC TGAGCGAGAC AAAGTGCACCCTGAAGTCCT TCACCGTGGA AAAGGGCATC TACCAGACCA GCAACTTCCG GGTGCAGCCCACCGAATCCA TCGTGCGGTT CCCCAATATC ACCAATCTGT GCCCCTTCGG CGAGGTGTTCAATGCCACCA GATTCGCCTC TGTGTACGCC TGGAACCGGA AGCGGATCAG CAATTGCGTGGCCGACTACT CCGTGCTGTA CAACTCCGCC AGCTTCAGCA CCTTCAAGTG CTACGGCGTGTCCCCTACCA AGCTGAACGA CCTGTGCTTC ACAAACGTGT ACGCCGACAG CTTCGTGATCCGGGGAGATG AAGTGCGGCA GATTGCCCCT GGACAGACAG GCAAGATCGC CGACTACAACTACAAGCTGC CCGACGACTT CACCGGCTGT GTGATTGCCT GGAACAGCAA CAACCTGGACTCCAAAGTCG GCGGCAACTA CAATTACAGG TACCGGCTGT TCCGGAAGTC CAATCTGAAGCCCTTCGAGC GGGACATCTC CACCGAGATC TATCAGGCCG GCAGCAAGCC TTGTAACGGCGTGGAAGGCT TCAACTGCTA CTTCCCACTG CAGTCCTACG GCTTTCAGCC CACAAATGGCGTGGGCTATC AGCCCTACAG AGTGGTGGTG CTGAGCTTCG AACTGCTGCA TGCCCCTGCCACAGTGTGCG GCCCTAAGAA AAGCACCAAT CTCGTGAAGA ACAAATGCGT GAACTTCAACTTCAACGGCC TGACCGGCAC CGGCGTGCTG ACAGAGAGCA ACAAGAAGTT CCTGCCATTCCAGCAGTTTG GCCGGGATAT CGCCGATACC ACAGACGCCG TTAGAGATCC CCAGACACTGGAAATCCTGG ACATCACCCC TTGCAGCTTC GGCGGAGTGT CTGTGATCAC CCCTGGCACCAACACCAGCA ATCAGGTGGC AGTGCTGTAC CAGGGCGTGA ACTGTACCGA AGTGCCCGTGGCCATTCACG CCGATCAGCT GACACCTACA TGGCGGGTGT ACTCCACCGG CAGCAATGTGTTTCAGACCA GAGCCGGCTG TCTGATCGGA GCCGAGCACG TGAACAATAG CTACGAGTGCGACATCCCCA TCGGCGCTGG AATCTGCGCC AGCTACCAGA CACAGACAAA CAGCAGGCGGAGAGCCAGAA GCGTGGCCAG CCAGAGCATC ATTGCCTACA CAATGTCTCT GGGCGCCGAGAACAGCGTGG CCTACTCCAA CAACTCTATC GCTATCCCCA CCAACTTCAC CATCAGCGTGACCACAGAGA TCCTGCCTGT GTCCATGACC AAGACCAGCG TGGACTGCAC CATGTACATCTGCGGCGATT CCACCGAGTG CTCCAACCTG CTGCTGCAGT ACGGCAGCTT CTGCACCCAGCTGAATAGAG CCCTGACAGG GATCGCCGTG GAACAGGACA AGAACACCCA AGAGGTGTTCGCCCAAGTGA AGCAGATCTA CAAGACCCCT CCTATCAAGG ACTTCGGCGG CTTCAATTTCAGCCAGATTC TGCCCGATCC TAGCAAGCCC AGCAAGCGGA GCTTCATCGA GGACCTGCTGTTCAACAAAG TGACACTGGC CGACGCCGGC TTCATCAAGC AGTATGGCGA TTGTCTGGGCGACATTGCCG CCAGGGATCT GATTTGCGCC CAGAAGTTTA ACGGACTGAC AGTGCTGCCTCCTCTGCTGA CCGATGAGAT GATCGCCCAG TACACATCTG CCCTGCTGGC CGGCACAATCACAAGCGGCT GGACATTTGG AGCAGGCGCC GCTCTGCAGA TCCCCTTTGC TATGCAGATGGCCTACCGGT TCAACGGCAT CGGAGTGACC CAGAATGTGC TGTACGAGAA CCAGAAGCTGATCGCCAACC AGTTCAACAG CGCCATCGGC AAGATCCAGG ACAGCCTGAG CAGCACAGCAAGCGCCCTGG GAAAGCTGCA GAACGTGGTC AACCAGAATG CCCAGGCACT GAACACCCTGGTCAAGCAGC TGTCCTCCAA CTTCGGCGCC ATCAGCTCTG TGCTGAACGA TATCCTGAGCAGACTGGACC CTCCTGAGGC CGAGGTGCAG ATCGACAGAC TGATCACAGG CAGACTGCAGAGCCTCCAGA CATACGTGAC CCAGCAGCTG ATCAGAGCCG CCGAGATTAG AGCCTCTGCCAATCTGGCCG CCACCAAGAT GTCTGAGTGT GTGCTGGGCC AGAGCAAGAG AGTGGACTTTTGCGGCAAGG GCTACCACCT GATGAGCTTC CCTCAGTCTG CCCCTCACGG CGTGGTGTTTCTGCACGTGA CATATGTGCC CGCTCAAGAG AAGAATTTCA CCACCGCTCC AGCCATCTGCCACGACGGCA AAGCCCACTT TCCTAGAGAA GGCGTGTTCG TGTCCAACGG CACCCATTGGTTCGTGACAC AGCGGAACTT CTACGAGCCC CAGATCATCA CCACCGACAA CACCTTCGTGTCTGGCAACT GCGACGTCGT GATCGGCATT GTGAACAATA CCGTGTACGA CCCTCTGCAGCCCGAGCTGG ACAGCTTCAA AGAGGAACTG GACAAGTACT TTAAGAACCA CACAAGCCCCGACGTGGACC TGGGCGATAT CAGCGGAATC AATGCCAGCG TCGTGAACAT CCAGAAAGAGATCGACCGGC TGAACGAGGT GGCCAAGAAT CTGAACGAGA GCCTGATCGA CCTGCAAGAACTGGGGAAGT ACGAGCAGTA CATCAAGTGG CCCTGGTACA TCTGGCTGGG CTTTATCGCCGGACTGATTG CCATCGTGAT GGTCACAATC ATGCTGTGTT GCATGACCAG CTGCTGTAGCTGCCTGAAGG GCTGTTGTAG CTGTGGCAGC TGCTGCAAGT TCGACGAGGA CGATTCTGAGCCCGTGCTGA AGGGCGTGAA ACTGCACTAC ACATGATGA TTTCACCTGG TACTGCATGC ACGCAATGCT AGCTGCCCCT TTCCCGTCCT GGGTACCCCGAGTCTCCCCC GACCTCGGGT CCCAGGTATG CTCCCACCTC CACCTGCCCC ACTCACCACCTCTGCTAGTT CCAGACACCT CCCAAGCACG CAGCAATGCA GCTCAAAACG CTTAGCCTAGCCACACCCCC ACGGGAAACA GCAGTGATTA ACCTTTAGCA ATAAACGAAA GTTTAACTAAGCTATACTAA CCCCAGGGTT GGTCAATTTC GTGCCAGCCA CACCCTGGAG CTAGCAAAAAAAAAA AAAAAAAAAA AAAAAAAAAA GCATATGACT AAAAAAAAAA AAAAAAAAAAAAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 157Full length sequence ofAGAAUAAACU AGUAUUCUUC UGGUCCCCAC AGACUCAGAG AGAACCCGCC ACCRBP020.14 (RNA)AUGUUCGUGU UCCUGGUGCU GCUGCCUCUG GUGUCCAGCC AGUGUGUGAA CCUGAGAACCAGAACACAGC UGCCUCCAGC CUACACCAAC AGCUUUACCA GAGGCGUGUA CUACCCCGACAAGGUGUUCA GAUCCAGCGU GCUGCACUCU ACCCAGGACC UGUUCCUGCC UUUCUUCAGCAACGUGACCU GGUUCCACGC CAUCCACGUG UCCGGCACCA AUGGCACCAA GAGAUUCGACAACCCCGUGC UGCCCUUCAA CGACGGGGUG UACUUUGCCA GCACCGAGAA GUCCAACAUCAUCAGAGGCU GGAUCUUCGG CACCACACUG GACAGCAAGA CCCAGAGCCU GCUGAUCGUGAACAACGCCA CCAACGUGGU CAUCAAAGUG UGCGAGUUCC AGUUCUGCAA CGACCCCUUCCUGGACGUCU ACUACCACAA GAACAACAAG AGCUGGAUGG AAAGCGGCGU GUACAGCAGCGCCAACAACU GCACCUUCGA GUACGUGUCC CAGCCUUUCC UGAUGGACCU GGAAGGCAAGCAGGGCAACU UCAAGAACCU GCGCGAGUUC GUGUUUAAGA ACAUCGACGG CUACUUCAAGAUCUACAGCA AGCACACCCC UAUCAACCUC GUGCGGGAUC UGCCUCAGGG CUUCUCUGCUCUGGAACCCC UGGUGGAUCU GCCCAUCGGC AUCAACAUCA CCCGGUUUCA GACACUGCUGGCCCUGCACA GAAGCUACCU GACACCUGGC GAUAGCAGCA GCGGAUGGAC AGCUGGUGCCGCCGCUUACU AUGUGGGCUA CCUGCAGCCU AGAACCUUCC UGCUGAAGUA CAACGAGAACGGCACCAUCA CCGACGCCGU GGAUUGUGCU CUGGAUCCUC UGAGCGAGAC AAAGUGCACCCUGAAGUCCU UCACCGUGGA AAAGGGCAUC UACCAGACCA GCAACUUCCG GGUGCAGCCCACCGAAUCCA UCGUGCGGUU CCCCAAUAUC ACCAAUCUGU GCCCCUUCGG CGAGGUGUUCAAUGCCACCA GAUUCGCCUC UGUGUACGCC UGGAACCGGA AGCGGAUCAG CAAUUGCGUGGCCGACUACU CCGUGCUGUA CAACUCCGCC AGCUUCAGCA CCUUCAAGUG CUACGGCGUGUCCCCUACCA AGCUGAACGA CCUGUGCUUC ACAAACGUGU ACGCCGACAG CUUCGUGAUCCGGGGAGAUG AAGUGCGGCA GAUUGCCCCU GGACAGACAG GCAAGAUCGC CGACUACAACUACAAGCUGC CCGACGACUU CACCGGCUGU GUGAUUGCCU GGAACAGCAA CAACCUGGACUCCAAAGUCG GCGGCAACUA CAAUUACAGG UACCGGCUGU UCCGGAAGUC CAAUCUGAAGCCCUUCGAGC GGGACAUCUC CACCGAGAUC UAUCAGGCCG GCAGCAAGCC UUGUAACGGCGUGGAAGGCU UCAACUGCUA CUUCCCACUG CAGUCCUACG GCUUUCAGCC CACAAAUGGCGUGGGCUAUC AGCCCUACAG AGUGGUGGUG CUGAGCUUCG AACUGCUGCA UGCCCCUGCCACAGUGUGCG GCCCUAAGAA AAGCACCAAU CUCGUGAAGA ACAAAUGCGU GAACUUCAACUUCAACGGCC UGACCGGCAC CGGCGUGCUG ACAGAGAGCA ACAAGAAGUU CCUGCCAUUCCAGCAGUUUG GCCGGGAUAU CGCCGAUACC ACAGACGCCG UUAGAGAUCC CCAGACACUGGAAAUCCUGG ACAUCACCCC UUGCAGCUUC GGCGGAGUGU CUGUGAUCAC CCCUGGCACCAACACCAGCA AUCAGGUGGC AGUGCUGUAC CAGGGCGUGA ACUGUACCGA AGUGCCCGUGGCCAUUCACG CCGAUCAGCU GACACCUACA UGGCGGGUGU ACUCCACCGG CAGCAAUGUGUUUCAGACCA GAGCCGGCUG UCUGAUCGGA GCCGAGCACG UGAACAAUAG CUACGAGUGCGACAUCCCCA UCGGCGCUGG AAUCUGCGCC AGCUACCAGA CACAGACAAA CAGCAGGCGGAGAGCCAGAA GCGUGGCCAG CCAGAGCAUC AUUGCCUACA CAAUGUCUCU GGGCGCCGAGAACAGCGUGG CCUACUCCAA CAACUCUAUC GCUAUCCCCA CCAACUUCAC CAUCAGCGUGACCACAGAGA UCCUGCCUGU GUCCAUGACC AAGACCAGCG UGGACUGCAC CAUGUACAUCUGCGGCGAUU CCACCGAGUG CUCCAACCUG CUGCUGCAGU ACGGCAGCUU CUGCACCCAGCUGAAUAGAG CCCUGACAGG GAUCGCCGUG GAACAGGACA AGAACACCCA AGAGGUGUUCGCCCAAGUGA AGCAGAUCUA CAAGACCCCU CCUAUCAAGG ACUUCGGCGG CUUCAAUUUCAGCCAGAUUC UGCCCGAUCC UAGCAAGCCC AGCAAGCGGA GCUUCAUCGA GGACCUGCUGUUCAACAAAG UGACACUGGC CGACGCCGGC UUCAUCAAGC AGUAUGGCGA UUGUCUGGGCGACAUUGCCG CCAGGGAUCU GAUUUGCGCC CAGAAGUUUA ACGGACUGAC AGUGCUGCCUCCUCUGCUGA CCGAUGAGAU GAUCGCCCAG UACACAUCUG CCCUGCUGGC CGGCACAAUCACAAGCGGCU GGACAUUUGG AGCAGGCGCC GCUCUGCAGA UCCCCUUUGC UAUGCAGAUGGCCUACCGGU UCAACGGCAU CGGAGUGACC CAGAAUGUGC UGUACGAGAA CCAGAAGCUGAUCGCCAACC AGUUCAACAG CGCCAUCGGC AAGAUCCAGG ACAGCCUGAG CAGCACAGCAAGCGCCCUGG GAAAGCUGCA GAACGUGGUC AACCAGAAUG CCCAGGCACU GAACACCCUGGUCAAGCAGC UGUCCUCCAA CUUCGGCGCC AUCAGCUCUG UGCUGAACGA UAUCCUGAGCAGACUGGACC CUCCUGAGGC CGAGGUGCAG AUCGACAGAC UGAUCACAGG CAGACUGCAGAGCCUCCAGA CAUACGUGAC CCAGCAGCUG AUCAGAGCCG CCGAGAUUAG AGCCUCUGCCAAUCUGGCCG CCACCAAGAU GUCUGAGUGU GUGCUGGGCC AGAGCAAGAG AGUGGACUUUUGCGGCAAGG GCUACCACCU GAUGAGCUUC CCUCAGUCUG CCCCUCACGG CGUGGUGUUUCUGCACGUGA CAUAUGUGCC CGCUCAAGAG AAGAAUUUCA CCACCGCUCC AGCCAUCUGCCACGACGGCA AAGCCCACUU UCCUAGAGAA GGCGUGUUCG UGUCCAACGG CACCCAUUGGUUCGUGACAC AGCGGAACUU CUACGAGCCC CAGAUCAUCA CCACCGACAA CACCUUCGUGUCUGGCAACU GCGACGUCGU GAUCGGCAUU GUGAACAAUA CCGUGUACGA CCCUCUGCAGCCCGAGCUGG ACAGCUUCAA AGAGGAACUG GACAAGUACU UUAAGAACCA CACAAGCCCCGACGUGGACC UGGGCGAUAU CAGCGGAAUC AAUGCCAGCG UCGUGAACAU CCAGAAAGAGAUCGACCGGC UGAACGAGGU GGCCAAGAAU CUGAACGAGA GCCUGAUCGA CCUGCAAGAACUGGGGAAGU ACGAGCAGUA CAUCAAGUGG CCCUGGUACA UCUGGCUGGG CUUUAUCGCCGGACUGAUUG CCAUCGUGAU GGUCACAAUC AUGCUGUGUU GCAUGACCAG CUGCUGUAGCUGCCUGAAGG GCUGUUGUAG CUGUGGCAGC UGCUGCAAGU UCGACGAGGA CGAUUCUGAGCCCGUGCUGA AGGGCGUGAA ACUGCACUAC ACAUGAUGAUUUCACCUGG UACUGCAUGC ACGCAAUGCU AGCUGCCCCU UUCCCGUCCU GGGUACCCCGAGUCUCCCCC GACCUCGGGU CCCAGGUAUG CUCCCACCUC CACCUGCCCC ACUCACCACCUCUGCUAGUU CCAGACACCU CCCAAGCACG CAGCAAUGCA GCUCAAAACG CUUAGCCUAGCCACACCCCC ACGGGAAACA GCAGUGAUUA ACCUUUAGCA AUAAACGAAA GUUUAACUAAGCUAUACUAA CCCCAGGGUU GGUCAAUUUC GUGCCAGCCA CACCCUGGAG CUAGC AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA GCAUAUGACU AAAAAAAAAA AAAAAAAAAAAAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA

TABLE 6 Sequences of RBP020.18 (Omicron BA.2-specific RNA vaccine) SEQID NO. Brief Description Sequence 64 Amino acid sequence of RNA-MFVFLVLLPLVSSQCVNLITRTQSYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHencoded SARS-CoV-2 S proteinVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFfrom an Omicron BA.2 variantCNDPFLDVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYF(with PRO mutations atKIYSKHTPINLGRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVpositions corresponding toGYLQPRTELLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITK986P and V987P of SEQ ID NO:NLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADS1; i.e., PRO mutations atFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVGGNYNYLYRLFRKSNLKPFEpositions 983 and 984 of RDISTEIYQAGNKPCNGVAGFNCYFPLRSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTSEQ ID NO: 64)NLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEYVNNSYECDIPIGAGICASYQTQTKSHRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLKRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKYFGGFNFSQILPDPSKPSKRSFIEDLLENKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRENGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNHNAQALNTLVKQLSSKFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWEVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT** 65 RNA sequence encoding a SARS-AUGUUCGUGUUCCUGGUGCUGCUGCCUCUGGUGUCCAGCCAGUGUGUGAACCUGAUCACCAGAACACoV-2 S protein from anCAGUCAUACACCAACAGCUUUACCAGAGGCGUGUACUACCCCGACAAGGUGUUCAGAUCCAGCGUGOmicron BA.2 variantCUGCACUCUACCCAGGACCUGUUCCUGCCUUUCUUCAGCAACGUGACCUGGUUCCACGCCAUCCACGUGUCCGGCACCAAUGGCACCAAGAGAUUCGACAACCCCGUGCUGCCCUUCAACGACGGGGUGUACUUUGCCAGCACCGAGAAGUCCAACAUCAUCAGAGGCUGGAUCUUCGGCACCACACUGGACAGCAAGACCCAGAGCCUGCUGAUCGUGAACAACGCCACCAACGUGGUCAUCAAAGUGUGCGAGUUCCAGUUCUGCAACGACCCCUUCCUGGACGUCUACUACCACAAGAACAACAAGAGCUGGAUGGAAAGCGAGUUCCGGGUGUACAGCAGCGCCAACAACUGCACCUUCGAGUACGUGUCCCAGCCUUUCCUGAUGGACCUGGAAGGCAAGCAGGGCAACUUCAAGAACCUGCGCGAGUUCGUGUUUAAGAACAUCGACGGCUACUUCAAGAUCUACAGCAAGCACACCCCUAUCAACCUCGGCCGGGAUCUGCCUCAGGGCUUCUCUGCUCUGGAACCCCUGGUGGAUCUGCCCAUCGGCAUCAACAUCACCCGGUUUCAGACACUGCUGGCCCUGCACAGAAGCUACCUGACACCUGGCGAUAGCAGCAGCGGAUGGACAGCUGGUGCCGCCGCUUACUAUGUGGGCUACCUGCAGCCUAGAACCUUCCUGCUGAAGUACAACGAGAACGGCACCAUCACCGACGCCGUGGAUUGUGCUCUGGAUCCUCUGAGCGAGACAAAGUGCACCCUGAAGUCCUUCACCGUGGAAAAGGGCAUCUACCAGACCAGCAACUUCCGGGUGCAGCCCACCGAAUCCAUCGUGCGGUUCCCCAAUAUCACCAAUCUGUGCCCCUUCGACGAGGUGUUCAAUGCCACCAGAUUCGCCUCUGUGUACGCCUGGAACCGGAAGCGGAUCAGCAAUUGCGUGGCCGACUACUCCGUGCUGUACAACUUCGCCCCCUUCUUCGCAUUCAAGUGCUACGGCGUGUCCCCUACCAAGCUGAACGACCUGUGCUUCACAAACGUGUACGCCGACAGCUUCGUGAUCCGGGGAAACGAAGUGUCACAGAUUGCCCCUGGACAGACAGGCAACAUCGCCGACUACAACUACAAGCUGCCCGACGACUUCACCGGCUGUGUGAUUGCCUGGAACAGCAACAAGCUGGACUCCAAAGUCGGCGGCAACUACAAUUACCUGUACCGGCUGUUCCGGAAGUCCAAUCUGAAGCCCUUCGAGCGGGACAUCUCCACCGAGAUCUAUCAGGCCGGCAACAAGCCUUGUAACGGCGUGGCAGGCUUCAACUGCUACUUCCCACUGAGGUCCUACGGCUUUAGGCCCACAUACGGCGUGGGCCACCAGCCCUACAGAGUGGUGGUGCUGAGCUUCGAACUGCUGCAUGCCCCUGCCACAGUGUGCGGCCCUAAGAAAAGCACCAAUCUCGUGAAGAACAAAUGCGUGAACUUCAACUUCAACGGCCUGACCGGCACCGGCGUGCUGACAGAGAGCAACAAGAAGUUCCUGCCAUUCCAGCAGUUUGGCCGGGAUAUCGCCGAUACCACAGACGCCGUUAGAGAUCCCCAGACACUGGAAAUCCUGGACAUCACCCCUUGCAGCUUCGGCGGAGUGUCUGUGAUCACCCCUGGCACCAACACCAGCAAUCAGGUGGCAGUGCUGUACCAGGGCGUGAACUGUACCGAAGUGCCCGUGGCCAUUCACGCCGAUCAGCUGACACCUACAUGGCGGGUGUACUCCACCGGCAGCAAUGUGUUUCAGACCAGAGCCGGCUGUCUGAUCGGAGCCGAGUACGUGAACAAUAGCUACGAGUGCGACAUCCCCAUCGGCGCUGGAAUCUGCGCCAGCUACCAGACACAGACAAAGAGCCACCGGAGAGCCAGAAGCGUGGCCAGCCAGAGCAUCAUUGCCUACACAAUGUCUCUGGGCGCCGAGAACAGCGUGGCCUACUCCAACAACUCUAUCGCUAUCCCCACCAACUUCACCAUCAGCGUGACCACAGAGAUCCUGCCUGUGUCCAUGACCAAGACCAGCGUGGACUGCACCAUGUACAUCUGCGGCGAUUCCACCGAGUGCUCCAACCUGCUGCUGCAGUACGGCAGCUUCUGCACCCAGCUGAAAAGAGCCCUGACAGGGAUCGCCGUGGAACAGGACAAGAACACCCAAGAGGUGUUCGCCCAAGUGAAGCAGAUCUACAAGACCCCUCCUAUCAAGUACUUCGGCGGCUUCAAUUUCAGCCAGAUUCUGCCCGAUCCUAGCAAGCCCAGCAAGCGGAGCUUCAUCGAGGACCUGCUGUUCAACAAAGUGACACUGGCCGACGCCGGCUUCAUCAAGCAGUAUGGCGAUUGUCUGGGCGACAUUGCCGCCAGGGAUCUGAUUUGCGCCCAGAAGUUUAACGGACUGACAGUGCUGCCUCCUCUGCUGACCGAUGAGAUGAUCGCCCAGUACACAUCUGCCCUGCUGGCCGGCACAAUCACAAGCGGCUGGACAUUUGGAGCAGGCGCCGCUCUGCAGAUCCCCUUUGCUAUGCAGAUGGCCUACCGGUUCAACGGCAUCGGAGUGACCCAGAAUGUGCUGUACGAGAACCAGAAGCUGAUCGCCAACCAGUUCAACAGCGCCAUCGGCAAGAUCCAGGACAGCCUGAGCAGCACAGCAAGCGCCCUGGGAAAGCUGCAGGACGUGGUCAACCACAAUGCCCAGGCACUGAACACCCUGGUCAAGCAGCUGUCCUCCAAGUUCGGCGCCAUCAGCUCUGUGCUGAACGAUAUCCUGAGCAGACUGGACCCUCCUGAGGCCGAGGUGCAGAUCGACAGACUGAUCACAGGCAGACUGCAGAGCCUCCAGACAUACGUGACCCAGCAGCUGAUCAGAGCCGCCGAGAUUAGAGCCUCUGCCAAUCUGGCCGCCACCAAGAUGUCUGAGUGUGUGCUGGGCCAGAGCAAGAGAGUGGACUUUUGCGGCAAGGGCUACCACCUGAUGAGCUUCCCUCAGUCUGCCCCUCACGGCGUGGUGUUUCUGCACGUGACAUAUGUGCCCGCUCAAGAGAAGAAUUUCACCACCGCUCCAGCCAUCUGCCACGACGGCAAAGCCCACUUUCCUAGAGAAGGCGUGUUCGUGUCCAACGGCACCCAUUGGUUCGUGACACAGCGGAACUUCUACGAGCCCCAGAUCAUCACCACCGACAACACCUUCGUGUCUGGCAACUGCGACGUCGUGAUCGGCAUUGUGAACAAUACCGUGUACGACCCUCUGCAGCCCGAGCUGGACAGCUUCAAAGAGGAACUGGACAAGUACUUUAAGAACCACACAAGCCCCGACGUGGACCUGGGCGAUAUCAGCGGAAUCAAUGCCAGCGUCGUGAACAUCCAGAAAGAGAUCGACCGGCUGAACGAGGUGGCCAAGAAUCUGAACGAGAGCCUGAUCGACCUGCAAGAACUGGGGAAGUACGAGCAGUACAUCAAGUGGCCCUGGUACAUCUGGCUGGGCUUUAUCGCCGGACUGAUUGCCAUCGUGAUGGUCACAAUCAUGCUGUGUUGCAUGACCAGCUGCUGUAGCUGCCUGAAGGGCUGUUGUAGCUGUGGCAGCUGCUCCAAGUUCGACGAGGACGAUUCUGAGCCCGUGCUGAAGGGCGUGAAACUGCACUACACAUGAUGA 66DNA sequence encoding aATGTTCGTGTTCCTGGTGCTGCTGCCTCTGGTGTCCAGCCAGTGTGTGAACCTGATCACCAGAACASARS-CoV-2 S protein fromCAGTCATACACCAACAGCTTTACCAGAGGCGTGTACTACCCCGACAAGGTGTTCAGATCCAGCGTGan Omicron BA.2 variantCTGCACTCTACCCAGGACCTGTTCCTGCCTTTCTTCAGCAACGTGACCTGGTTCCACGCCATCCACGTGTCCGGCACCAATGGCACCAAGAGATTCGACAACCCCGTGCTGCCCTTCAACGACGGGGTGTACTTTGCCAGCACCGAGAAGTCCAACATCATCAGAGGCTGGATCTTCGGCACCACACTGGACAGCAAGACCCAGAGCCTGCTGATCGTGAACAACGCCACCAACGTGGTCATCAAAGTGTGCGAGTTCCAGTTCTGCAACGACCCCTTCCTGGACGTCTACTACCACAAGAACAACAAGAGCTGGATGGAAAGCGAGTTCCGGGTGTACAGCAGCGCCAACAACTGCACCTTCGAGTACGTGTCCCAGCCTTTCCTGATGGACCTGGAAGGCAAGCAGGGCAACTTCAAGAACCTGCGCGAGTTCGTGTTTAAGAACATCGACGGCTACTTCAAGATCTACAGCAAGCACACCCCTATCAACCTCGGCCGGGATCTGCCTCAGGGCTTCTCTGCTCTGGAACCCCTGGTGGATCTGCCCATCGGCATCAACATCACCCGGTTTCAGACACTGCTGGCCCTGCACAGAAGCTACCTGACACCTGGCGATAGCAGCAGCGGATGGACAGCTGGTGCCGCCGCTTACTATGTGGGCTACCTGCAGCCTAGAACCTTCCTGCTGAAGTACAACGAGAACGGCACCATCACCGACGCCGTGGATTGTGCTCTGGATCCTCTGAGCGAGACAAAGTGCACCCTGAAGTCCTTCACCGTGGAAAAGGGCATCTACCAGACCAGCAACTTCCGGGTGCAGCCCACCGAATCCATCGTGCGGTTCCCCAATATCACCAATCTGTGCCCCTTCGACGAGGTGTTCAATGCCACCAGATTCGCCTCTGTGTACGCCTGGAACCGGAAGCGGATCAGCAATTGCGTGGCCGACTACTCCGTGCTGTACAACTTCGCCCCCTTCTTCGCATTCAAGTGCTACGGCGTGTCCCCTACCAAGCTGAACGACCTGTGCTTCACAAACGTGTACGCCGACAGCTTCGTGATCCGGGGAAACGAAGTGTCACAGATTGCCCCTGGACAGACAGGCAACATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGTGTGATTGCCTGGAACAGCAACAAGCTGGACTCCAAAGTCGGCGGCAACTACAATTACCTGTACCGGCTGTTCCGGAAGTCCAATCTGAAGCCCTTCGAGCGGGACATCTCCACCGAGATCTATCAGGCCGGCAACAAGCCTTGTAACGGCGTGGCAGGCTTCAACTGCTACTTCCCACTGAGGTCCTACGGCTTTAGGCCCACATACGGCGTGGGCCACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAACTGCTGCATGCCCCTGCCACAGTGTGCGGCCCTAAGAAAAGCACCAATCTCGTGAAGAACAAATGCGTGAACTTCAACTTCAACGGCCTGACCGGCACCGGCGTGCTGACAGAGAGCAACAAGAAGTTCCTGCCATTCCAGCAGTTTGGCCGGGATATCGCCGATACCACAGACGCCGTTAGAGATCCCCAGACACTGGAAATCCTGGACATCACCCCTTGCAGCTTCGGCGGAGTGTCTGTGATCACCCCTGGCACCAACACCAGCAATCAGGTGGCAGTGCTGTACCAGGGCGTGAACTGTACCGAAGTGCCCGTGGCCATTCACGCCGATCAGCTGACACCTACATGGCGGGTGTACTCCACCGGCAGCAATGTGTTTCAGACCAGAGCCGGCTGTCTGATCGGAGCCGAGTACGTGAACAATAGCTACGAGTGCGACATCCCCATCGGCGCTGGAATCTGCGCCAGCTACCAGACACAGACAAAGAGCCACCGGAGAGCCAGAAGCGTGGCCAGCCAGAGCATCATTGCCTACACAATGTCTCTGGGCGCCGAGAACAGCGTGGCCTACTCCAACAACTCTATCGCTATCCCCACCAACTTCACCATCAGCGTGACCACAGAGATCCTGCCTGTGTCCATGACCAAGACCAGCGTGGACTGCACCATGTACATCTGCGGCGATTCCACCGAGTGCTCCAACCTGCTGCTGCAGTACGGCAGCTTCTGCACCCAGCTGAAAAGAGCCCTGACAGGGATCGCCGTGGAACAGGACAAGAACACCCAAGAGGTGTTCGCCCAAGTGAAGCAGATCTACAAGACCCCTCCTATCAAGTACTTCGGCGGCTTCAATTTCAGCCAGATTCTGCCCGATCCTAGCAAGCCCAGCAAGCGGAGCTTCATCGAGGACCTGCTGTTCAACAAAGTGACACTGGCCGACGCCGGCTTCATCAAGCAGTATGGCGATTGTCTGGGCGACATTGCCGCCAGGGATCTGATTTGCGCCCAGAAGTTTAACGGACTGACAGTGCTGCCTCCTCTGCTGACCGATGAGATGATCGCCCAGTACACATCTGCCCTGCTGGCCGGCACAATCACAAGCGGCTGGACATTTGGAGCAGGCGCCGCTCTGCAGATCCCCTTTGCTATGCAGATGGCCTACCGGTTCAACGGCATCGGAGTGACCCAGAATGTGCTGTACGAGAACCAGAAGCTGATCGCCAACCAGTTCAACAGCGCCATCGGCAAGATCCAGGACAGCCTGAGCAGCACAGCAAGCGCCCTGGGAAAGCTGCAGGACGTGGTCAACCACAATGCCCAGGCACTGAACACCCTGGTCAAGCAGCTGTCCTCCAAGTTCGGCGCCATCAGCTCTGTGCTGAACGATATCCTGAGCAGACTGGACCCTCCTGAGGCCGAGGTGCAGATCGACAGACTGATCACAGGCAGACTGCAGAGCCTCCAGACATACGTGACCCAGCAGCTGATCAGAGCCGCCGAGATTAGAGCCTCTGCCAATCTGGCCGCCACCAAGATGTCTGAGTGTGTGCTGGGCCAGAGCAAGAGAGTGGACTTTTGCGGCAAGGGCTACCACCTGATGAGCTTCCCTCAGTCTGCCCCTCACGGCGTGGTGTTTCTGCACGTGACATATGTGCCCGCTCAAGAGAAGAATTTCACCACCGCTCCAGCCATCTGCCACGACGGCAAAGCCCACTTTCCTAGAGAAGGCGTGTTCGTGTCCAACGGCACCCATTGGTTCGTGACACAGCGGAACTTCTACGAGCCCCAGATCATCACCACCGACAACACCTTCGTGTCTGGCAACTGCGACGTCGTGATCGGCATTGTGAACAATACCGTGTACGACCCTCTGCAGCCCGAGCTGGACAGCTTCAAAGAGGAACTGGACAAGTACTTTAAGAACCACACAAGCCCCGACGTGGACCTGGGCGATATCAGCGGAATCAATGCCAGCGTCGTGAACATCCAGAAAGAGATCGACCGGCTGAACGAGGTGGCCAAGAATCTGAACGAGAGCCTGATCGACCTGCAAGAACTGGGGAAGTACGAGCAGTACATCAAGTGGCCCTGGTACATCIGGCTGGGCTTTATCGCCGGACTGATTGCCATCGTGATGGTCACAATCATGCTGTGTTGCATGACCAGCTGCTGTAGCTGCCTGAAGGGCTGTTGTAGCTGTGGCAGCTGCTGCAAGTTCGACGAGGACGATTCTGAGCCCGTGCTGAAGGGCGTGAAACTGCACTACACATGATGA 67Full length RNA constructAGAAUAAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGUUCGUGUUCCsequence of RBP020.18UGGUGCUGCUGCCUCUGGUGUCCAGCCAGUGUGUGAACCUGAUCACCAGAACACAGUCAUACACCAACAGCUUUACCAGAGGCGUGUACUACCCCGACAAGGUGUUCAGAUCCAGCGUGCUGCACUCUACCCAGGACCUGUUCCUGCCUUUCUUCAGCAACGUGACCUGGUUCCACGCCAUCCACGUGUCCGGCACCAAUGGCACCAAGAGAUUCGACAACCCCGUGCUGCCCUUCAACGACGGGGUGUACUUUGCCAGCACCGAGAAGUCCAACAUCAUCAGAGGCUGGAUCUUCGGCACCACACUGGACAGCAAGACCCAGAGCCUGCUGAUCGUGAACAACGCCACCAACGUGGUCAUCAAAGUGUGCGAGUUCCAGUUCUGCAACGACCCCUUCCUGGACGUCUACUACCACAAGAACAACAAGAGCUGGAUGGAAAGCGAGUUCCGGGUGUACAGCAGCGCCAACAACUGCACCUUCGAGUACGUGUCCCAGCCUUUCCUGAUGGACCUGGAAGGCAAGCAGGGCAACUUCAAGAACCUGCGCGAGUUCGUGUUUAAGAACAUCGACGGCUACUUCAAGAUCUACAGCAAGCACACCCCUAUCAACCUCGGCCGGGAUCUGCCUCAGGGCUUCUCUGCUCUGGAACCCCUGGUGGAUCUGCCCAUCGGCAUCAACAUCACCCGGUUUCAGACACUGCUGGCCCUGCACAGAAGCUACCUGACACCUGGCGAUAGCAGCAGCGGAUGGACAGCUGGUGCCGCCGCUUACUAUGUGGGCUACCUGCAGCCUAGAACCUUCCUGCUGAAGUACAACGAGAACGGCACCAUCACCGACGCCGUGGAUUGUGCUCUGGAUCCUCUGAGCGAGACAAAGUGCACCCUGAAGUCCUUCACCGUGGAAAAGGGCAUCUACCAGACCAGCAACUUCCGGGUGCAGCCCACCGAAUCCAUCGUGCGGUUCCCCAAUAUCACCAAUCUGUGCCCCUUCGACGAGGUGUUCAAUGCCACCAGAUUCGCCUCUGUGUACGCCUGGAACCGGAAGCGGAUCAGCAAUUGCGUGGCCGACUACUCCGUGCUGUACAACUUCGCCCCCUUCUUCGCAUUCAAGUGCUACGGCGUGUCCCCUACCAAGCUGAACGACCUGUGCUUCACAAACGUGUACGCCGACAGCUUCGUGAUCCGGGGAAACGAAGUGUCACAGAUUGCCCCUGGACAGACAGGCAACAUCGCCGACUACAACUACAAGCUGCCCGACGACUUCACCGGCUGUGUGAUUGCCUGGAACAGCAACAAGCUGGACUCCAAAGUCGGCGGCAACUACAAUUACCUGUACCGGCUGUUCCGGAAGUCCAAUCUGAAGCCCUUCGAGCGGGACAUCUCCACCGAGAUCUAUCAGGCCGGCAACAAGCCUUGUAACGGCGUGGCAGGCUUCAACUGCUACUUCCCACUGAGGUCCUACGGCUUUAGGCCCACAUACGGCGUGGGCCACCAGCCCUACAGAGUGGUGGUGCUGAGCUUCGAACUGCUGCAUGCCCCUGCCACAGUGUGCGGCCCUAAGAAAAGCACCAAUCUCGUGAAGAACAAAUGCGUGAACUUCAACUUCAACGGCCUGACCGGCACCGGCGUGCUGACAGAGAGCAACAAGAAGUUCCUGCCAUUCCAGCAGUUUGGCCGGGAUAUCGCCGAUACCACAGACGCCGUUAGAGAUCCCCAGACACUGGAAAUCCUGGACAUCACCCCUUGCAGCUUCGGCGGAGUGUCUGUGAUCACCCCUGGCACCAACACCAGCAAUCAGGUGGCAGUGCUGUACCAGGGCGUGAACUGUACCGAAGUGCCCGUGGCCAUUCACGCCGAUCAGCUGACACCUACAUGGCGGGUGUACUCCACCGGCAGCAAUGUGUUUCAGACCAGAGCCGGCUGUCUGAUCGGAGCCGAGUACGUGAACAAUAGCUACGAGUGCGACAUCCCCAUCGGCGCUGGAAUCUGCGCCAGCUACCAGACACAGACAAAGAGCCACCGGAGAGCCAGAAGCGUGGCCAGCCAGAGCAUCAUUGCCUACACAAUGUCUCUGGGCGCCGAGAACAGCGUGGCCUACUCCAACAACUCUAUCGCUAUCCCCACCAACUUCACCAUCAGCGUGACCACAGAGAUCCUGCCUGUGUCCAUGACCAAGACCAGCGUGGACUGCACCAUGUACAUCUGCGGCGAUUCCACCGAGUGCUCCAACCUGCUGCUGCAGUACGGCAGCUUCUGCACCCAGCUGAAAAGAGCCCUGACAGGGAUCGCCGUGGAACAGGACAAGAACACCCAAGAGGUGUUCGCCCAAGUGAAGCAGAUCUACAAGACCCCUCCUAUCAAGUACUUCGGCGGCUUCAAUUUCAGCCAGAUUCUGCCCGAUCCUAGCAAGCCCAGCAAGCGGAGCUUCAUCGAGGACCUGCUGUUCAACAAAGUGACACUGGCCGACGCCGGCUUCAUCAAGCAGUAUGGCGAUUGUCUGGGCGACAUUGCCGCCAGGGAUCUGAUUUGCGCCCAGAAGUUUAACGGACUGACAGUGCUGCCUCCUCUGCUGACCGAUGAGAUGAUCGCCCAGUACACAUCUGCCCUGCUGGCCGGCACAAUCACAAGCGGCUGGACAUUUGGAGCAGGCGCCGCUCUGCAGAUCCCCUUUGCUAUGCAGAUGGCCUACCGGUUCAACGGCAUCGGAGUGACCCAGAAUGUGCUGUACGAGAACCAGAAGCUGAUCGCCAACCAGUUCAACAGCGCCAUCGGCAAGAUCCAGGACAGCCUGAGCAGCACAGCAAGCGCCCUGGGAAAGCUGCAGGACGUGGUCAACCACAAUGCCCAGGCACUGAACACCCUGGUCAAGCAGCUGUCCUCCAAGUUCGGCGCCAUCAGCUCUGUGCUGAACGAUAUCCUGAGCAGACUGGACCCUCCUGAGGCCGAGGUGCAGAUCGACAGACUGAUCACAGGCAGACUGCAGAGCCUCCAGACAUACGUGACCCAGCAGCUGAUCAGAGCCGCCGAGAUUAGAGCCUCUGCCAAUCUGGCCGCCACCAAGAUGUCUGAGUGUGUGCUGGGCCAGAGCAAGAGAGUGGACUUUUGCGGCAAGGGCUACCACCUGAUGAGCUUCCCUCAGUCUGCCCCUCACGGCGUGGUGUUUCUGCACGUGACAUAUGUGCCCGCUCAAGAGAAGAAUUUCACCACCGCUCCAGCCAUCUGCCACGACGGCAAAGCCCACUUUCCUAGAGAAGGCGUGUUCGUGUCCAACGGCACCCAUUGGUUCGUGACACAGCGGAACUUCUACGAGCCCCAGAUCAUCACCACCGACAACACCUUCGUGUCUGGCAACUGCGACGUCGUGAUCGGCAUUGUGAACAAUACCGUGUACGACCCUCUGCAGCCCGAGCUGGACAGCUUCAAAGAGGAACUGGACAAGUACUUUAAGAACCACACAAGCCCCGACGUGGACCUGGGCGAUAUCAGCGGAAUCAAUGCCAGCGUCGUGAACAUCCAGAAAGAGAUCGACCGGCUGAACGAGGUGGCCAAGAAUCUGAACGAGAGCCUGAUCGACCUGCAAGAACUGGGGAAGUACGAGCAGUACAUCAAGUGGCCCUGGUACAUCUGGCUGGGCUUUAUCGCCGGACUGAUUGCCAUCGUGAUGGUCACAAUCAUGCUGUGUUGCAUGACCAGCUGCUGUAGCUGCCUGAAGGGCUGUUGUAGCUGUGGCAGCUGCUGCAAGUUCGACGAGGACGAUUCUGAGCCCGUGCUGAAGGGCGUGAAACUGCACUACACAUGAUGACUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUGGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 68Full length DNA constructAGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGTTCGTGTTCCsequence of RBP020.18TGGTGCTGCTGCCTCTGGTGTCCAGCCAGTGTGTGAACCTGATCACCAGAACACAGTCATACACCAACAGCTTTACCAGAGGCGTGTACTACCCCGACAAGGTGTTCAGATCCAGCGTGCTGCACTCTACCCAGGACCTGTTCCTGCCTTTCTTCAGCAACGTGACCTGGTTCCACGCCATCCACGTGTCCGGCACCAATGGCACCAAGAGATTCGACAACCCCGTGCTGCCCTTCAACGACGGGGTGTACTTTGCCAGCACCGAGAAGTCCAACATCATCAGAGGCTGGATCTTCGGCACCACACTGGACAGCAAGACCCAGAGCCTGCTGATCGTGAACAACGCCACCAACGTGGTCATCAAAGTGTGCGAGTTCCAGTTCTGCAACGACCCCTTCCTGGACGTCTACTACCACAAGAACAACAAGAGCTGGATGGAAAGCGAGTTCCGGGTGTACAGCAGCGCCAACAACTGCACCTTCGAGTACGTGTCCCAGCCTTTCCTGATGGACCTGGAAGGCAAGCAGGGCAACTTCAAGAACCTGCGCGAGTTCGTGTTTAAGAACATCGACGGCTACTTCAAGATCTACAGCAAGCACACCCCTATCAACCTCGGCCGGGATCTGCCTCAGGGCTTCTCTGCTCTGGAACCCCTGGTGGATCTGCCCATCGGCATCAACATCACCCGGTTTCAGACACTGCTGGCCCTGCACAGAAGCTACCTGACACCTGGCGATAGCAGCAGCGGATGGACAGCTGGTGCCGCCGCTTACTATGTGGGCTACCTGCAGCCTAGAACCTTCCTGCTGAAGTACAACGAGAACGGCACCATCACCGACGCCGTGGATTGTGCTCTGGATCCTCTGAGCGAGACAAAGTGCACCCTGAAGTCCTTCACCGTGGAAAAGGGCATCTACCAGACCAGCAACTTCCGGGTGCAGCCCACCGAATCCATCGTGCGGTTCCCCAATATCACCAATCTGTGCCCCTTCGACGAGGTGTTCAATGCCACCAGATTCGCCTCTGTGTACGCCTGGAACCGGAAGCGGATCAGCAATTGCGTGGCCGACTACTCCGTGCTGTACAACTTCGCCCCCTTCTTCGCATTCAAGTGCTACGGCGTGTCCCCTACCAAGCTGAACGACCTGTGCTTCACAAACGTGTACGCCGACAGCTTCGTGATCCGGGGAAACGAAGTGTCACAGATTGCCCCTGGACAGACAGGCAACATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGTGTGATTGCCTGGAACAGCAACAAGCTGGACTCCAAAGTCGGCGGCAACTACAATTACCTGTACCGGCTGTTCCGGAAGTCCAATCTGAAGCCCTTCGAGCGGGACATCTCCACCGAGATCTATCAGGCCGGCAACAAGCCTTGTAACGGCGTGGCAGGCTTCAACTGCTACTTCCCACTGAGGTCCTACGGCTTTAGGCCCACATACGGCGTGGGCCACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAACTGCTGCATGCCCCTGCCACAGTGTGCGGCCCTAAGAAAAGCACCAATCTCGTGAAGAACAAATGCGTGAACTTCAACTTCAACGGCCTGACCGGCACCGGCGTGCTGACAGAGAGCAACAAGAAGTTCCTGCCATTCCAGCAGTTIGGCCGGGATATCGCCGATACCACAGACGCCGTTAGAGATCCCCAGACACTGGAAATCCTGGACATCACCCCTTGCAGCTTCGGCGGAGTGTCTGTGATCACCCCIGGCACCAACACCAGCAATCAGGTGGCAGTGCTGTACCAGGGCGTGAACTGTACCGAAGTGCCCGTGGCCATTCACGCCGATCAGCTGACACCTACATGGCGGGTGTACTCCACCGGCAGCAATGTGTTTCAGACCAGAGCCGGCTGTCTGATCGGAGCCGAGTACGTGAACAATAGCTACGAGTGCGACATCCCCATCGGCGCTGGAATCTGCGCCAGCTACCAGACACAGACAAAGAGCCACCGGAGAGCCAGAAGCGTGGCCAGCCAGAGCATCATTGCCTACACAATGTCTCTGGGCGCCGAGAACAGCGTGGCCTACTCCAACAACTCTATCGCTATCCCCACCAACTTCACCATCAGCGTGACCACAGAGATCCTGCCTGTGTCCATGACCAAGACCAGCGTGGACTGCACCATGTACATCTGCGGCGATTCCACCGAGTGCTCCAACCTGCTGCTGCAGTACGGCAGCTTCTGCACCCAGCTGAAAAGAGCCCTGACAGGGATCGCCGTGGAACAGGACAAGAACACCCAAGAGGTGTTCGCCCAAGTGAAGCAGATCTACAAGACCCCTCCTATCAAGTACTTCGGCGGCTTCAATTTCAGCCAGATTCTGCCCGATCCTAGCAAGCCCAGCAAGCGGAGCTTCATCGAGGACCTGCTGTTCAACAAAGTGACACTGGCCGACGCCGGCTTCATCAAGCAGTATGGCGATTGTCTGGGCGACATTGCCGCCAGGGATCTGATTTGCGCCCAGAAGTTTAACGGACTGACAGTGCTGCCTCCTCTGCTGACCGATGAGATGATCGCCCAGTACACATCTGCCCTGCTGGCCGGCACAATCACAAGCGGCTGGACATTTGGAGCAGGCGCCGCTCTGCAGATCCCCTTTGCTATGCAGATGGCCTACCGGTTCAACGGCATCGGAGTGACCCAGAATGTGCTGTACGAGAACCAGAAGCTGATCGCCAACCAGTTCAACAGCGCCATCGGCAAGATCCAGGACAGCCTGAGCAGCACAGCAAGCGCCCTGGGAAAGCTGCAGGACGTGGTCAACCACAATGCCCAGGCACTGAACACCCTGGTCAAGCAGCTGTCCTCCAAGTTCGGCGCCATCAGCTCTGTGCTGAACGATATCCTGAGCAGACTGGACCCTCCTGAGGCCGAGGTGCAGATCGACAGACTGATCACAGGCAGACTGCAGAGCCTCCAGACATACGTGACCCAGCAGCTGATCAGAGCCGCCGAGATTAGAGCCTCTGCCAATCTGGCCGCCACCAAGATGTCTGAGTGTGTGCTGGGCCAGAGCAAGAGAGTGGACTTTTGCGGCAAGGGCTACCACCTGATGAGCTTCCCTCAGTCTGCCCCTCACGGCGTGGTGTTTCTGCACGTGACATATGTGCCCGCTCAAGAGAAGAATTTCACCACCGCTCCAGCCATCTGCCACGACGGCAAAGCCCACTTTCCTAGAGAAGGCGTGTTCGTGTCCAACGGCACCCATTGGTTCGTGACACAGCGGAACTTCTACGAGCCCCAGATCATCACCACCGACAACACCTTCGTGTCTGGCAACTGCGACGTCGTGATCGGCATTGTGAACAATACCGTGTACGACCCTCTGCAGCCCGAGCTGGACAGCTTCAAAGAGGAACTGGACAAGTACTTTAAGAACCACACAAGCCCCGACGTGGACCTGGGCGATATCAGCGGAATCAATGCCAGCGTCGTGAACATCCAGAAAGAGATCGACCGGCTGAACGAGGTGGCCAAGAATCTGAACGAGAGCCTGATCGACCTGCAAGAACTGGGGAAGTACGAGCAGTACATCAAGTGGCCCTGGTACATCTGGCTGGGCTTTATCGCCGGACTGATTGCCATCGTGATGGTCACAATCATGCTGTGTTGCATGACCAGCTGCTGTAGCTGCCTGAAGGGCTGTTGTAGCTGTGGCAGCTGCTGCAAGTTCGACGAGGACGATTCTGAGCCCGTGCTGAAGGGCGTGAAACTGCACTACACATGATGACTCGAGCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTGGAGCTAGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

TABLE 7 Description of sequences of RBP020.18(Omicron BA.2-specific RNA vaccine)as described in Table 6 above (amino acid mutationpositioning shown relative to SEQ ID NO: 1) Construct Omicron BA.2 P2Antigen P2-mutated full spike protein Changes Amino Acid^(a)mRNA Nucleotides (location) Furin site RRAR (SEQ CGGAGAGCCAGAID NO: 136) (SEQ ID NO: 137) (2088-2099) Proline K986P CCU (3000-3002)V987P CCU (3003-3005) Lineage T19I AUC (108-110) L24del / P25del /P26del / A27S UCA (123-125) G142D GAC (468-470) V213G GGC (681-683)G339D GAC (1059-1061) S371F UUC (1155-1157) S373P CCC (1161-1163) S375FUUC (1167-1169) T376A GCA (1170-1172) D405N AAC (1257-1259) R408SUCA (1266-1268) K417N AAC (1293-1295) N440K AAG (1362-1364) S477NAAC (1473-1475) T478K AAG (1476-1478) E484A GCA (1494-1496) Q493RAGG (1521-1523) Q498R AGG (1536-1538) N501Y UAC (1545-1547) Y505HCAC (1557-1559) D614G GGC (1884-1886) H655Y UAC (2007-2009) N679KAAG (2079-2081) P681H CAC (2085-2087) N764K AAA (2334-2336) D796YUAC (2430-2432) Q954H CAC (2904-2906) N969K AAG (2949-2951)

TABLE 8 Sequences of RBP020.22 (Omicron BA.4/BA.5-specific RNA vaccine)SEQ ID NO. Brief Description Sequence 69 Amino acid sequence of RNA-MEVELVLLPLVSSQCVNLITRTQSYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAISencoded SARS-CoV-2 S proteinGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNfrom an Omicron BA.4/BA.5DPFLDVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIvariant (with PRO mutations atYSKHTPINLGRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYpositions corresponding toLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLpositions K986P and V987P ofCPFDEVENATRFASVYAWNRKRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVSEQ ID NO: 7, i.e., atIRGNEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVGGNYNYRYRLFRKSNLKPFERDresidues 981 and 982 ofISTEIYQAGNKPCNGVAGVNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLSEQ ID NO: 69)VKNKCVNFNFNGLTGTGVLTESNKKELPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEYVNNSYECDIPIGAGICASYQTQTKSHRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLKRALTGIAVEQDKNTQEVFAQVKQLYKTPPIKYFGGFNFSQILPDPSKPSKRSFIEDLLENKVTLADAGFIKQYGDCLGDIAARDLICAQKENGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRENGIGVTQNVLYENQKLIANQENSAIGKIQDSLSSTASALGKLQDVVNHNAQALNTLVKQLSSKFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKEDEDDSEPVLKGVKLHYT** 70 RNA sequence encoding a SARS-AUGUUCGUGUUCCUGGUGCUGCUGCCUCUGGUGUCCAGCCAGUGUGUGAACCUGAUCACCAGAACACoV-2 S protein from anCAGUCAUACACCAACAGCUUUACCAGAGGCGUGUACUACCCCGACAAGGUGUUCAGAUCCAGCGUGOmicron BA.4/BA.5 variantCUGCACUCUACCCAGGACCUGUUCCUGCCUUUCUUCAGCAACGUGACCUGGUUCCACGCCAUCUCCGGCACCAAUGGCACCAAGAGAUUCGACAACCCCGUGCUGCCCUUCAACGACGGGGUGUACUUUGCCAGCACCGAGAAGUCCAACAUCAUCAGAGGCUGGAUCUUCGGCACCACACUGGACAGCAAGACCCAGAGCCUGCUGAUCGUGAACAACGCCACCAACGUGGUCAUCAAAGUGUGCGAGUUCCAGUUCUGCAACGACCCCUUCCUGGACGUCUACUACCACAAGAACAACAAGAGCUGGAUGGAAAGCGAGUUCCGGGUGUACAGCAGCGCCAACAACUGCACCUUCGAGUACGUGUCCCAGCCUCUCCUGAUGGACCUGGAAGGCAAGCAGGGCAACUUCAAGAACCUGCGCGAGUUCGUGUUUAAGAACAUCGACGGCUACUUCAAGAUCUACAGCAAGCACACCCCUAUCAACCUCGGCCGGGAUCUGCCUCAGGGCUUCUCUGCUCUGGAACCCCUGGUGGAUCUGCCCAUCGGCAUCAACAUCACCCGGUUUCAGACACUGCUGGCCCUGCACAGAAGCUACCUGACACCUGGCGAUAGCAGCAGCGGAUGGACAGCUGGUGCCGCCGCUUACUAUGUGGGCUACCUGCAGCCUAGAACCUUCCUGCUGAAGUACAACGAGAACGGCACCAUCACCGACGCCGUGGAUUGUGCUCUGGAUCCUCUGAGCGAGACAAAGUGCACCCUGAAGUCCUUCACCGUGGAAAAGGGCAUCUACCAGACCAGCAACUUCCGGGUGCAGCCCACCGAAUCCAUCGUGCGGUUCCCCAAUAUCACCAAUCUGUGCCCCUUCGACGAGGUGUUCAAUGCCACCAGAUUCGCCUCUGUGUACGCCUGGAACCGGAAGCGGAUCAGCAAUUGCGUGGCCGACUACUCCGUGCUGUACAACUUCGCCCCCUUCUUCGCAUUCAAGUGCUACGGCGUGUCCCCUACCAAGCUGAACGACCUGUGCUUCACAAACGUGUACGCCGACAGCUUCGUGAUCCGGGGAAACGAAGUGCGGCAGAUUGCCCCUGGACAGACAGGCAACAUCGCCGACUACAACUACAAGCUGCCCGACGACUUCACCGGCUGUGUGAUUGCCUGGAACAGCAACAAGCUGGACUCCAAAGUCGGCGGCAACUACAAUUACAGGUACCGGCUGUUCCGGAAGUCCAAUCUGAAGCCCUUCGAGCGGGACAUCUCCACCGAGAUCUAUCAGGCCGGCAACAAGCCUUGUAACGGCGUGGCAGGCGUGAACUGCUACUUCCCACUGCAGUCCUACGGCUUUAGGCCCACAUACGGCGUGGGCCACCAGCCCUACAGAGUGGUGGUGCUGAGCUUCGAACUGCUGCAUGCCCCUGCCACAGUGUGCGGCCCUAAGAAAAGCACCAAUCUCGUGAAGAACAAAUGCGUGAACUUCAACUUCAACGGCCUGACCGGCACCGGCGUGCUGACAGAGAGCAACAAGAAGUUCCUGCCAUUCCAGCAGUUUGGCCGGGAUAUCGCCGAUACCACAGACGCCGUUAGAGAUCCCCAGACACUGGAAAUCCUGGACAUCACCCCUUGCAGCUUCGGCGGAGUGUCUGUGAUCACCCCUGGCACCAACACCAGCAAUCAGGUGGCAGUGCUGUACCAGGGCGUGAACUGUACCGAAGUGCCCGUGGCCAUUCACGCCGAUCAGCUGACACCUACAUGGCGGGUGUACUCCACCGGCAGCAAUGUGUUUCAGACCAGAGCCGGCUGUCUGAUCGGAGCCGAGUACGUGAACAAUAGCUACGAGUGCGACAUCCCCAUCGGCGCUGGAAUCUGCGCCAGCUACCAGACACAGACAAAGAGCCACCGGAGAGCCAGAAGCGUGGCCAGCCAGAGCAUCAUUGCCUACACAAUGUCUCUGGGCGCCGAGAACAGCGUGGCCUACUCCAACAACUCUAUCGCUAUCCCCACCAACUUCACCAUCAGCGUGACCACAGAGAUCCUGCCUGUGUCCAUGACCAAGACCAGCGUGGACUGCACCAUGUACAUCUGCGGCGAUUCCACCGAGUGCUCCAACCUGCUGCUGCAGUACGGCAGCUUCUGCACCCAGCUGAAAAGAGCCCUGACAGGGAUCGCCGUGGAACAGGACAAGAACACCCAAGAGGUGUUCGCCCAAGUGAAGCAGAUCUACAAGACCCCUCCUAUCAAGUACUUCGGCGGCUUCAAUUUCAGCCAGAUUCUGCCCGAUCCUAGCAAGCCCAGCAAGCGGAGCUUCAUCGAGGACCUGCUGUUCAACAAAGUGACACUGGCCGACGCCGGCUUCAUCAAGCAGUAUGGCGAUUGUCUGGGCGACAUUGCCGCCAGGGAUCUGAUUUGCGCCCAGAAGUUUAACGGACUGACAGUGCUGCCUCCUCUGCUGACCGAUGAGAUGAUCGCCCAGUACACAUCUGCCCUGCUGGCCGGCACAAUCACAAGCGGCUGGACAUUUGGAGCAGGCGCCGCUCUGCAGAUCCCCUUUGCUAUGCAGAUGGCCUACCGGUUCAACGGCAUCGGAGUGACCCAGAAUGUGCUGUACGAGAACCAGAAGCUGAUCGCCAACCAGUUCAACAGCGCCAUCGGCAAGAUCCAGGACAGCCUGAGCAGCACAGCAAGCGCCCUGGGAAAGCUGCAGGACGUGGUCAACCACAAUGCCCAGGCACUGAACACCCUGGUCAAGCAGCUGUCCUCCAAGUUCGGCGCCAUCAGCUCUGUGCUGAACGAUAUCCUGAGCAGACUGGACCCUCCUGAGGCCGAGGUGCAGAUCGACAGACUGAUCACAGGCAGACUGCAGAGCCUCCAGACAUACGUGACCCAGCAGCUGAUCAGAGCCGCCGAGAUUAGAGCCUCUGCCAAUCUGGCCGCCACCAAGAUGUCUGAGUGUGUGCUGGGCCAGAGCAAGAGAGUGGACUUUUGCGGCAAGGGCUACCACCUGAUGAGCUUCCCUCAGUCUGCCCCUCACGGCGUGGUGUUUCUGCACGUGACAUAUGUGCCCGCUCAAGAGAAGAAUUUCACCACCGCUCCAGCCAUCUGCCACGACGGCAAAGCCCACUUUCCUAGAGAAGGCGUGUUCGUGUCCAACGGCACCCAUUGGUUCGUGACACAGCGGAACUUCUACGAGCCCCAGAUCAUCACCACCGACAACACCUUCGUGUCUGGCAACUGCGACGUCGUGAUCGGCAUUGUGAACAAUACCGUGUACGACCCUCUGCAGCCCGAGCUGGACAGCUUCAAAGAGGAACUGGACAAGUACUUUAAGAACCACACAAGCCCCGACGUGGACCUGGGCGAUAUCAGCGGAAUCAAUGCCAGCGUCGUGAACAUCCAGAAAGAGAUCGACCGGCUGAACGAGGUGGCCAAGAAUCUGAACGAGAGCCUGAUCGACCUGCAAGAACUGGGGAAGUACGAGCAGUACAUCAAGUGGCCCUGGUACAUCUGGCUGGGCUUUAUCGCCGGACUGAUUGCCAUCGUGAUGGUCACAAUCAUGCUGUGUUGCAUGACCAGCUGCUGUAGCUGCCUGAAGGGCUGUUGUAGCUGUGGCAGCUGCUGCAAGUUCGACGAGGACGAUUCUGAGCCCGUGCUGAAGGGCGUGAAACUGCACUACACAUGAUGA 71DNA sequence encoding a SARS-ATGTTCGTGTTCCTGGTGCTGCTGCCTCTGGTGTCCAGCCAGTGTGTGAACCTGATCACCAGAACACoV-2 S protein from anCAGTCATACACCAACAGCITTACCAGAGGCGTGTACTACCCCGACAAGGTGTTCAGATCCAGCGTGOmicron BA.4/BA.5 variantCTGCACTCTACCCAGGACCTGTTCCTGCCTTTCTTCAGCAACGTGACCTGGTTCCACGCCATCTCCGGCACCAATGGCACCAAGAGATTCGACAACCCCGTGCTGCCCTTCAACGACGGGGTGTACTTTGCCAGCACCGAGAAGTCCAACATCATCAGAGGCTGGATCTTCGGCACCACACTGGACAGCAAGACCCAGAGCCTGCTGATCGTGAACAACGCCACCAACGTGGTCATCAAAGTGTGCGAGTTCCAGTTCTGCAACGACCCCTTCCTGGACGTCTACTACCACAAGAACAACAAGAGCTGGATGGAAAGCGAGTTCCGGGTGTACAGCAGCGCCAACAACTGCACCTTCGAGTACGTGTCCCAGCCTTTCCTGATGGACCTGGAAGGCAAGCAGGGCAACTTCAAGAACCTGCGCGAGTTCGTGTTTAAGAACATCGACGGCTACTTCAAGATCTACAGCAAGCACACCCCTATCAACCTCGGCCGGGATCTGCCTCAGGGCTTCTCTGCTCTGGAACCCCTGGTGGATCTGCCCATCGGCATCAACATCACCCGGTTTCAGACACTGCTGGCCCTGCACAGAAGCTACCTGACACCTGGCGATAGCAGCAGCGGATGGACAGCTGGTGCCGCCGCTTACTATGTGGGCTACCTGCAGCCTAGAACCTTCCTGCTGAAGTACAACGAGAACGGCACCATCACCGACGCCGTGGATTGTGCTCTGGATCCTCTGAGCGAGACAAAGTGCACCCTGAAGTCCTTCACCGTGGAAAAGGGCATCTACCAGACCAGCAACTTCCGGGTGCAGCCCACCGAATCCATCGTGCGGTTCCCCAATATCACCAATCTGTGCCCCTTCGACGAGGTGTTCAATGCCACCAGATTCGCCTCTGTGTACGCCTGGAACCGGAAGCGGATCAGCAATTGCGTGGCCGACTACTCCGTGCTGTACAACTTCGCCCCCTTCTTCGCATTCAAGTGCTACGGCGTGTCCCCTACCAAGCTGAACGACCTGTGCTTCACAAACGTGTACGCCGACAGCTTCGTGATCCGGGGAAACGAAGTGCGGCAGATTGCCCCTGGACAGACAGGCAACATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGTGTGATTGCCTGGAACAGCAACAAGCTGGACTCCAAAGTCGGCGGCAACTACAATTACAGGTACCGGCTGTTCCGGAAGTCCAATCTGAAGCCCTTCGAGCGGGACATCTCCACCGAGATCTATCAGGCCGGCAACAAGCCTTGTAACGGCGTGGCAGGCGTGAACTGCTACTTCCCACTGCAGTCCTACGGCTTTAGGCCCACATACGGCGTGGGCCACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAACTGCTGCATGCCCCTGCCACAGTGTGCGGCCCTAAGAAAAGCACCAATCTCGTGAAGAACAAATGCGTGAACTTCAACTTCAACGGCCTGACCGGCACCGGCGTGCTGACAGAGAGCAACAAGAAGTTCCTGCCATTCCAGCAGTTTGGCCGGGATATCGCCGATACCACAGACGCCGTTAGAGATCCCCAGACACTGGAAATCCTGGACATCACCCCTTGCAGCTTCGGCGGAGTGTCTGTGATCACCCCTGGCACCAACACCAGCAATCAGGTGGCAGTGCTGTACCAGGGCGTGAACTGTACCGAAGTGCCCGTGGCCATTCACGCCGATCAGCTGACACCTACATGGGGGGTGTACTCCACCGGCAGCAATGTGITTCAGACCAGAGCCGGCTGTCTGATCGGAGCCGAGTACGTGAACAATAGCTACGAGTGCGACATCCCCATCGGCGCTGGAATCTGCGCCAGCTACCAGACACAGACAAAGAGCCACCGGAGAGCCAGAAGCGTGGCCAGCCAGAGCATCATTGCCTACACAATGTCTCTGGGCGCCGAGAACAGCGTGGCCTACTCCAACAACTCTATCGCTATCCCCACCAACTTCACCATCAGCGTGACCACAGAGATCCTGCCTGTGTCCATGACCAAGACCAGCGTGGACTGCACCATGTACATCTGCGGCGATTCCACCGAGTGCTCCAACCTGCTGCTGCAGTACGGCAGCTTCTGCACCCAGCTGAAAAGAGCCCTGACAGGGATCGCCGTGGAACAGGACAAGAACACCCAAGAGGTGTTCGCCCAAGTGAAGCAGATCTACAAGACCCCTCCTATCAAGTACTTCGGCGGCTTCAATTTCAGCCAGATTCTGCCCGATCCTAGCAAGCCCAGCAAGCGGAGCTTCATCGAGGACCTGCTGTTCAACAAAGTGACACTGGCCGACGCCGGCTTCATCAAGCAGTATGGCGATTGTCTGGGCGACATTGCCGCCAGGGATCTGATTTGCGCCCAGAAGTTTAACGGACTGACAGTGCTGCCTCCTCTGCTGACCGATGAGATGATCGCCCAGTACACATCTGCCCTGCTGGCCGGCACAATCACAAGCGGCTGGACATTTGGAGCAGGCGCCGCTCTGCAGATCCCCTTTGCTATGCAGATGGCCTACCGGTTCAACGGCATCGGAGTGACCCAGAATGTGCTGTACGAGAACCAGAAGCTGATCGCCAACCAGTTCAACAGCGCCATCGGCAAGATCCAGGACAGCCTGAGCAGCACAGCAAGCGCCCTGGGAAAGCTGCAGGACGTGGTCAACCACAATGCCCAGGCACTGAACACCCTGGTCAAGCAGCTGTCCTCCAAGTTCGGCGCCATCAGCTCTGTGCTGAACGATATCCTGAGCAGACTGGACCCTCCTGAGGCCGAGGTGCAGATCGACAGACTGATCACAGGCAGACTGCAGAGCCTCCAGACATACGTGACCCAGCAGCTGATCAGAGCCGCCGAGATTAGAGCCTCTGCCAATCTGGCCGCCACCAAGATGTCTGAGTGTGTGCTGGGCCAGAGCAAGAGAGTGGACTTTTGCGGCAAGGGCTACCACCTGATGAGCTTCCCTCAGTCTGCCCCTCACGGCGTGGTGTTTCTGCACGTGACATATGTGCCCGCTCAAGAGAAGAATTTCACCACCGCTCCAGCCATCTGCCACGACGGCAAAGCCCACTTTCCTAGAGAAGGCGTGTTCGTGTCCAACGGCACCCATTGGTTCGTGACACAGCGGAACTTCTACGAGCCCCAGATCATCACCACCGACAACACCTTCGTGTCTGGCAACTGCGACGTCGTGATCGGCATTGTGAACAATACCGTGTACGACCCTCTGCAGCCCGAGCTGGACAGCTTCAAAGAGGAACTGGACAAGTACTTTAAGAACCACACAAGCCCCGACGTGGACCTGGGCGATATCAGCGGAATCAATGCCAGCGTCGTGAACATCCAGAAAGAGATCGACCGGCTGAACGAGGTGGCCAAGAATCTGAACGAGAGCCTGATCGACCTGCAAGAACTGGGGAAGTACGAGCAGTACATCAAGTGGCCCTGGTACATCTGGCTGGGCTTTATCGCCGGACTGATTGCCATCGTGATGGTCACAATCATGCTGTGTTGCATGACCAGCTGCTGTAGCTGCCTGAAGGGCTGTTGTAGCTGTGGCAGCTGCTGCAAGTTCGACGAGGACGATTCTGAGCCCGTGCTGAAGGGCGTGAAACTGCACTACACATGATGA 72Full length RNA constructAGAAUAAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGUUCGUGUUCCsequence of RBP020.22UGGUGCUGCUGCCUCUGGUGUCCAGCCAGUGUGUGAACCUGAUCACCAGAACACAGUCAUACACCAACAGCUUUACCAGAGGCGUGUACUACCCCGACAAGGUGUUCAGAUCCAGCGUGCUGCACUCUACCCAGGACCUGUUCCUGCCUUUCUUCAGCAACGUGACCUGGUUCCACGCCAUCUCCGGCACCAAUGGCACCAAGAGAUUCGACAACCCCGUGCUGCCCUUCAACGACGGGGUGUACUUUGCCAGCACCGAGAAGUCCAACAUCAUCAGAGGCUGGAUCUUCGGCACCACACUGGACAGCAAGACCCAGAGCCUGCUGAUCGUGAACAACGCCACCAACGUGGUCAUCAAAGUGUGCGAGUUCCAGUUCUGCAACGACCCCUUCCUGGACGUCUACUACCACAAGAACAACAAGAGCUGGAUGGAAAGCGAGUUCCGGGUGUACAGCAGCGCCAACAACUGCACCUUCGAGUACGUGUCCCAGCCUUUCCUGAUGGACCUGGAAGGCAAGCAGGGCAACUUCAAGAACCUGCGCGAGUUCGUGUUUAAGAACAUCGACGGCUACUUCAAGAUCUACAGCAAGCACACCCCUAUCAACCUCGGCCGGGAUCUGCCUCAGGGCUUCUCUGCUCUGGAACCCCUGGUGGAUCUGCCCAUCGGCAUCAACAUCACCCGGUUUCAGACACUGCUGGCCCUGCACAGAAGCUACCUGACACCUGGCGAUAGCAGCAGCGGAUGGACAGCUGGUGCCGCCGCUUACUAUGUGGGCUACCUGCAGCCUAGAACCUUCCUGCUGAAGUACAACGAGAACGGCACCAUCACCGACGCCGUGGAUUGUGCUCUGGAUCCUCUGAGCGAGACAAAGUGCACCCUGAAGUCCUUCACCGUGGAAAAGGGCAUCUACCAGACCAGCAACUUCCGGGUGCAGCCCACCGAAUCCAUCGUGCGGUUCCCCAAUAUCACCAAUCUGUGCCCCUUCGACGAGGUGUUCAAUGCCACCAGAUUCGCCUCUGUGUACGCCUGGAACCGGAAGCGGAUCAGCAAUUGCGUGGCCGACUACUCCGUGCUGUACAACUUCGCCCCCUUCUUCGCAUUCAAGUGCUACGGCGUGUCCCCUACCAAGCUGAACGACCUGUGCUUCACAAACGUGUACGCCGACAGCUUCGUGAUCCGGGGAAACGAAGUGCGGCAGAUUGCCCCUGGACAGACAGGCAACAUCGCCGACUACAACUACAAGCUGCCCGACGACUUCACCGGCUGUGUGAUUGCCUGGAACAGCAACAAGCUGGACUCCAAAGUCGGCGGCAACUACAAUUACAGGUACCGGCUGUUCCGGAAGUCCAAUCUGAAGCCCUUCGAGCGGGACAUCUCCACCGAGAUCUAUCAGGCCGGCAACAAGCCUUGUAACGGCGUGGCAGGCGUGAACUGCUACUUCCCACUGCAGUCCUACGGCUUUAGGCCCACAUACGGCGUGGGCCACCAGCCCUACAGAGUGGUGGUGCUGAGCUUCGAACUGCUGCAUGCCCCUGCCACAGUGUGCGGCCCUAAGAAAAGCACCAAUCUCGUGAAGAACAAAUGCGUGAACUUCAACUUCAACGGCCUGACCGGCACCGGCGUGCUGACAGAGAGCAACAAGAAGUUCCUGCCAUUCCAGCAGUUUGGCCGGGAUAUCGCCGAUACCACAGACGCCGUUAGAGAUCCCCAGACACUGGAAAUCCUGGACAUCACCCCUUGCAGCUUCGGCGGAGUGUCUGUGAUCACCCCUGGCACCAACACCAGCAAUCAGGUGGCAGUGCUGUACCAGGGCGUGAACUGUACCGAAGUGCCCGUGGCCAUUCACGCCGAUCAGCUGACACCUACAUGGCGGGUGUACUCCACCGGCAGCAAUGUGUUUCAGACCAGAGCCGGCUGUCUGAUCGGAGCCGAGUACGUGAACAAUAGCUACGAGUGCGACAUCCCCAUCGGCGCUGGAAUCUGCGCCAGCUACCAGACACAGACAAAGAGCCACCGGAGAGCCAGAAGCGUGGCCAGCCAGAGCAUCAUUGCCUACACAAUGUCUCUGGGCGCCGAGAACAGCGUGGCCUACUCCAACAACUCUAUCGCUAUCCCCACCAACUUCACCAUCAGCGUGACCACAGAGAUCCUGCCUGUGUCCAUGACCAAGACCAGCGUGGACUGCACCAUGUACAUCUGCGGCGAUUCCACCGAGUGCUCCAACCUGCUGCUGCAGUACGGCAGCUUCUGCACCCAGCUGAAAAGAGCCCUGACAGGGAUCGCCGUGGAACAGGACAAGAACACCCAAGAGGUGUUCGCCCAAGUGAAGCAGAUCUACAAGACCCCUCCUAUCAAGUACUUCGGCGGCUUCAAUUUCAGCCAGAUUCUGCCCGAUCCUAGCAAGCCCAGCAAGCGGAGCUUCAUCGAGGACCUGCUGUUCAACAAAGUGACACUGGCCGACGCCGGCUUCAUCAAGCAGUAUGGCGAUUGUCUGGGCGACAUUGCCGCCAGGGAUCUGAUUUGCGCCCAGAAGUUUAACGGACUGACAGUGCUGCCUCCUCUGCUGACCGAUGAGAUGAUCGCCCAGUACACAUCUGCCCUGCUGGCCGGCACAAUCACAAGCGGCUGGACAUUUGGAGCAGGCGCCGCUCUGCAGAUCCCCUUUGCUAUGCAGAUGGCCUACCGGUUCAACGGCAUCGGAGUGACCCAGAAUGUGCUGUACGAGAACCAGAAGCUGAUCGCCAACCAGUUCAACAGCGCCAUCGGCAAGAUCCAGGACAGCCUGAGCAGCACAGCAAGCGCCCUGGGAAAGCUGCAGGACGUGGUCAACCACAAUGCCCAGGCACUGAACACCCUGGUCAAGCAGCUGUCCUCCAAGUUCGGCGCCAUCAGCUCUGUGCUGAACGAUAUCCUGAGCAGACUGGACCCUCCUGAGGCCGAGGUGCAGAUCGACAGACUGAUCACAGGCAGACUGCAGAGCCUCCAGACAUACGUGACCCAGCAGCUGAUCAGAGCCGCCGAGAUUAGAGCCUCUGCCAAUCUGGCCGCCACCAAGAUGUCUGAGUGUGUGCUGGGCCAGAGCAAGAGAGUGGACUUUUGCGGCAAGGGCUACCACCUGAUGAGCUUCCCUCAGUCUGCCCCUCACGGCGUGGUGUUUCUGCACGUGACAUAUGUGCCCGCUCAAGAGAAGAAUUUCACCACCGCUCCAGCCAUCUGCCACGACGGCAAAGCCCACUUUCCUAGAGAAGGCGUGUUCGUGUCCAACGGCACCCAUUGGUUCGUGACACAGCGGAACUUCUACGAGCCCCAGAUCAUCACCACCGACAACACCUUCGUGUCUGGCAACUGCGACGUCGUGAUCGGCAUUGUGAACAAUACCGUGUACGACCCUCUGCAGCCCGAGCUGGACAGCUUCAAAGAGGAACUGGACAAGUACUUUAAGAACCACACAAGCCCCGACGUGGACCUGGGCGAUAUCAGCGGAAUCAAUGCCAGCGUCGUGAACAUCCAGAAAGAGAUCGACCGGCUGAACGAGGUGGCCAAGAAUCUGAACGAGAGCCUGAUCGACCUGCAAGAACUGGGGAAGUACGAGCAGUACAUCAAGUGGCCCUGGUACAUCUGGCUGGGCUUUAUCGCCGGACUGAUUGCCAUCGUGAUGGUCACAAUCAUGCUGUGUUGCAUGACCAGCUGCUGUAGCUGCCUGAAGGGCUGUUGUAGCUGUGGCAGCUGCUGCAAGUUCGACGAGGACGAUUCUGAGCCCGUGCUGAAGGGCGUGAAACUGCACUACACAUGAUGACUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUGGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 73Full length DNA constructAGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGTTCGTGTTCCsequence of RBP020.22TGGTGCTGCTGCCTCTGGTGTCCAGCCAGTGTGTGAACCTGATCACCAGAACACAGTCATACACCAACAGCTTTACCAGAGGCGTGTACTACCCCGACAAGGTGTTCAGATCCAGCGTGCTGCACTCTACCCAGGACCTGTTCCTGCCTTTCTTCAGCAACGTGACCTGGTTCCACGCCATCTCCGGCACCAATGGCACCAAGAGATTCGACAACCCCGTGCTGCCCTTCAACGACGGGGTGTACTTTGCCAGCACCGAGAAGTCCAACATCATCAGAGGCTGGATCTTCGGCACCACACTGGACAGCAAGACCCAGAGCCTGCTGATCGTGAACAACGCCACCAACGTGGTCATCAAAGTGTGCGAGTTCCAGTTCTGCAACGACCCCTTCCTGGACGTCTACTACCACAAGAACAACAAGAGCTGGATGGAAAGCGAGTTCCGGGTGTACAGCAGCGCCAACAACTGCACCTTCGAGTACGTGTCCCAGCCTTTCCTGATGGACCTGGAAGGCAAGCAGGGCAACTTCAAGAACCTGCGCGAGTTCGTGTTTAAGAACATCGACGGCTACTTCAAGATCTACAGCAAGCACACCCCTATCAACCTCGGCCGGGATCTGCCTCAGGGCTTCTCTGCTCTGGAACCCCTGGTGGATCTGCCCATCGGCATCAACATCACCCGGTTTCAGACACTGCTGGCCCTGCACAGAAGCTACCTGACACCTGGCGATAGCAGCAGCGGATGGACAGCTGGTGCCGCCGCTTACTATGTGGGCTACCTGCAGCCTAGAACCTTCCTGCTGAAGTACAACGAGAACGGCACCATCACCGACGCCGTGGATTGTGCTCTGGATCCTCTGAGCGAGACAAAGTGCACCCTGAAGTCCTTCACCGTGGAAAAGGGCATCTACCAGACCAGCAACTTCCGGGTGCAGCCCACCGAATCCATCGTGCGGTTCCCCAATATCACCAATCTGTGCCCCTTCGACGAGGTGTTCAATGCCACCAGATTCGCCTCTGTGTACGCCTGGAACCGGAAGCGGATCAGCAATTGCGTGGCCGACTACTCCGTGCTGTACAACTTCGCCCCCTTCTTCGCATTCAAGTGCTACGGCGTGTCCCCTACCAAGCTGAACGACCTGTGCTTCACAAACGTGTACGCCGACAGCTTCGTGATCCGGGGAAACGAAGTGCGGCAGATTGCCCCTGGACAGACAGGCAACATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGTGTGATTGCCTGGAACAGCAACAAGCTGGACTCCAAAGTCGGCGGCAACTACAATTACAGGTACCGGCTGTTCCGGAAGTCCAATCTGAAGCCCTTCGAGCGGGACATCTCCACCGAGATCTATCAGGCCGGCAACAAGCCTTGTAACGGCGTGGCAGGCGTGAACTGCTACTTCCCACTGCAGTCCTACGGCTTTAGGCCCACATACGGCGTGGGCCACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAACTGCTGCATGCCCCTGCCACAGTGTGCGGCCCTAAGAAAAGCACCAATCTCGTGAAGAACAAATGCGTGAACTTCAACTTCAACGGCCTGACCGGCACCGGCGTGCTGACAGAGAGCAACAAGAAGTTCCTGCCATTCCAGCAGTTTGGCCGGGATATCGCCGATACCACAGACGCCGTTAGAGATCCCCAGACACTGGAAATCCTGGACATCACCCCTTGCAGCTTCGGCGGAGTGTCTGTGATCACCCCTGGCACCAACACCAGCAATCAGGTGGCAGTGCTGTACCAGGGCGTGAACTGTACCGAAGTGCCCGTGGCCATTCACGCCGATCAGCTGACACCTACATGGCGGGTGTACTCCACCGGCAGCAATGTGTTTCAGACCAGAGCCGGCTGTCTGATCGGAGCCGAGTACGTGAACAATAGCTACGAGTGCGACATCCCCATCGGCGCTGGAATCTGCGCCAGCTACCAGACACAGACAAAGAGCCACCGGAGAGCCAGAAGCGTGGCCAGCCAGAGCATCATTGCCTACACAATGTCTCTGGGCGCCGAGAACAGCGTGGCCTACTCCAACAACTCTATCGCTATCCCCACCAACTTCACCATCAGCGTGACCACAGAGATCCTGCCTGTGTCCATGACCAAGACCAGCGTGGACTGCACCATGTACATCTGCGGCGATTCCACCGAGTGCTCCAACCTGCTGCTGCAGTACGGCAGCTTCTGCACCCAGCTGAAAAGAGCCCTGACAGGGATCGCCGTGGAACAGGACAAGAACACCCAAGAGGTGTTCGCCCAAGTGAAGCAGATCTACAAGACCCCTCCTATCAAGTACTTCGGCGGCTTCAATTTCAGCCAGATTCTGCCCGATCCTAGCAAGCCCAGCAAGCGGAGCTTCATCGAGGACCTGCTGTTCAACAAAGTGACACTGGCCGACGCCGGCTTCATCAAGCAGTATGGCGATTGTCTGGGCGACATTGCCGCCAGGGATCTGATTTGCGCCCAGAAGTTTAACGGACTGACAGTGCTGCCTCCTCTGCTGACCGATGAGATGATCGCCCAGTACACATCTGCCCTGCTGGCCGGCACAATCACAAGCGGCTGGACATTTGGAGCAGGCGCCGCTCTGCAGATCCCCTTTGCTATGCAGATGGCCTACCGGTTCAACGGCATCGGAGTGACCCAGAATGTGCTGTACGAGAACCAGAAGCTGATCGCCAACCAGTTCAACAGCGCCATCGGCAAGATCCAGGACAGCCTGAGCAGCACAGCAAGCGCCCTGGGAAAGCTGCAGGACGTGGTCAACCACAATGCCCAGGCACTGAACACCCTGGTCAAGCAGCTGTCCTCCAAGTTCGGCGCCATCAGCTCTGTGCTGAACGATATCCTGAGCAGACTGGACCCTCCTGAGGCCGAGGTGCAGATCGACAGACTGATCACAGGCAGACTGCAGAGCCTCCAGACATACGTGACCCAGCAGCTGATCAGAGCCGCCGAGATTAGAGCCTCTGCCAATCTGGCCGCCACCAAGATGTCTGAGTGTGTGCTGGGCCAGAGCAAGAGAGTGGACTTTTGCGGCAAGGGCTACCACCTGATGAGCTTCCCTCAGTCTGCCCCTCACGGCGTGGTGTTTCTGCACGTGACATATGTGCCCGCTCAAGAGAAGAATTTCACCACCGCTCCAGCCATCTGCCACGACGGCAAAGCCCACTTTCCTAGAGAAGGCGTGTTCGTGTCCAACGGCACCCATTGGTTCGTGACACAGCGGAACTTCTACGAGCCCCAGATCATCACCACCGACAACACCTTCGTGTCTGGCAACTGCGACGTCGTGATCGGCATTGTGAACAATACCGTGTACGACCCTCTGCAGCCCGAGCTGGACAGCTTCAAAGAGGAACTGGACAAGTACTTTAAGAACCACACAAGCCCCGACGTGGACCTGGGCGATATCAGCGGAATCAATGCCAGCGTCGTGAACATCCAGAAAGAGATCGACCGGCTGAACGAGGTGGCCAAGAATCTGAACGAGAGCCTGATCGACCTGCAAGAACTGGGGAAGTACGAGCAGTACATCAAGTGGCCCTGGTACATCTGGCTGGGCTTTATCGCCGGACTGATTGCCATCGTGATGGTCACAATCATGCTGTGTTGCATGACCAGCTGCTGTAGCTGCCTGAAGGGCTGTTGTAGCTGTGGCAGCTGCTGCAAGTTCGACGAGGACGATTCTGAGCCCGTGCTGAAGGGCGTGAAACTGCACTACACATGATGACTCGAGCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTGGAGCTAGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

TABLE 9Description of RBP020.22 (Omicron BA.4/BA.5-specific RNA vaccine)as shown in Table 8 above (positioning of amino acid mutationsshown relative to SEQ ID NO: 1) Construct Omicron BA.4/BA.5 P2 AntigenP2-mutated full spike protein Changes Amino Acid^(a)mRNA Nucleotides (location) Furin site RRAR(SEQ ID CGGAGAGCCAGA NO: 136)(SEQ ID NO: 137) (2082-2093) Proline K986P CCU (3000-3002) V987PCCU (3003-3005) Lineage T191 AUC (108-110) L24del / P25del / P26del /A27S UCA (123-125) H69del / V70del / G142D GAC (462-464) V213GGGC (675-677) G339D GAC (1053-1055) S371F UUC (1149-1151) S373PCCC (1155-1157) S375F UUC (1161-1163) T376A GCA (1164-1166) D405NAAC (1251-1253) K417N AAC (1287-1289) N440K AAG (1356-1358) L452RAGG (1392-1394) S477N AAC (1467-1469) T478K AAG (1470-1472) E484AGCA (1488-1490) F486V GUG (1494-1496) Q498R AGG (1530-1532) N501YUAC (1539-1541) Y505H CAC (1551-1553) D614G GGC (1878-1880) H655YUAC (2001-2003) N679K AAG (2073-2075) P681H CAC (2079-2081) N764KAAA (2328-2330) D796Y UAC (2424-2426) Q954H CAC (2898-2900) N969KAAG (2943-2945)

TABLE 10Sequence of one embodiment of Omicron BA.4/BA.5-specific RNA vaccine SEQID NO. Brief Description Sequence 74 Amino acid sequence of RNA-MFVFLVLLPLVSSQCVNLITRTQSYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAISencoded SARS-CoV-2 S proteinGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNfrom an Omicron BA.4/BA.5DPFLDVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIvariant (with PRO mutationsYSKHTPINLGRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYat positions corresponding toLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLK986P and V987P of SEQ ID NO:CPFDEVENATRFASVYAWNRKRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFV1; i.e., PRO mutations atIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVGGNYNYRYRLFRKSNLKPFERDpositions 981 and 982 of SEQISTEIYQAGNKPCNGVAGVNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLID NO: 74)VKNKCVNFNFNGLTGTGVLTESNKKELPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEYVNNSYECDIPIGAGICASYQTQTKSHRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNETISVTTEILPVSMTKTSVDCTMYICGDSTECSNELLQYGSFCTQLKRALTGIAVEQDKNTQEVFAQVKQLYKTPPIKYFGGFNFSQILPDPSKPSKRSFIEDLLENKVTLADAGFIKQYGDCLGDIAARDLICAQKENGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRENGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNHNAQALNTLVKQLSSKFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNETTAPAICHDGKAHFPREGVEVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT 75 RNA sequence encoding aAUGUUCGUGUUCCUGGUGCUGCUGCCUCUGGUGUCCAGCCAGUGUGUGAACCUGAUCACCAGAACASARS-CoV-2 S protein from anCAGAGCUACACCAACAGCUUUACCAGAGGCGUGUACUACCCCGACAAGGUGUUCAGAUCCAGCGUGOmicron BA.4/BA.5 variantCUGCACUCUACCCAGGACCUGUUCCUGCCUUUCUUCAGCAACGUGACCUGGUUCCACGCCAUCUCC(with proline residues atGGCACCAAUGGCACCAAGAGAUUCGACAACCCCGUGCUGCCCUUCAACGACGGGGUGUACUUUGCCpositions corresponding toAGCACCGAGAAGUCCAACAUCAUCAGAGGCUGGAUCUUCGGCACCACACUGGACAGCAAGACCCAGK986P and V987P of SEQ ID NO:AGCCUGCUGAUCGUGAACAACGCCACCAACGUGGUCAUCAAAGUGUGCGAGUUCCAGUUCUGCAAC 1)GACCCCUUCCUGGACGUGUACUACCACAAGAACAACAAGAGCUGGAUGGAAAGCGAGUUCCGGGUGUACAGCAGCGCCAACAACUGCACCUUCGAGUACGUGUCCCAGCCUUUCCUGAUGGACCUGGAAGGCAAGCAGGGCAACUUCAAGAACCUGCGCGAGUUCGUGUUUAAGAACAUCGACGGCUACUUCAAGAUCUACAGCAAGCACACCCCUAUCAACCUCGGCCGGGAUCUGCCUCAGGGCUUCUCUGCUCUGGAACCCCUGGUGGAUCUGCCCAUCGGCAUCAACAUCACCCGGUUUCAGACACUGCUGGCCCUGCACAGAAGCUACCUGACACCUGGCGAUAGCAGCAGCGGAUGGACAGCUGGUGCCGCCGCUUACUAUGUGGGCUACCUGCAGCCUAGAACCUUCCUGCUGAAGUACAACGAGAACGGCACCAUCACCGACGCCGUGGAUUGUGCUCUGGAUCCUCUGAGCGAGACAAAGUGCACCCUGAAGUCCUUCACCGUGGAAAAGGGCAUCUACCAGACCAGCAACUUCCGGGUGCAGCCCACCGAAUCCAUCGUGCGGUUCCCCAAUAUCACCAAUCUGUGCCCCUUCGACGAGGUGUUCAAUGCCACCAGAUUCGCCUCUGUGUACGCCUGGAACCGGAAGCGGAUCAGCAAUUGCGUGGCCGACUACUCCGUGCUGUACAACUUCGCCCCCUUCUUCGCCUUCAAGUGCUACGGCGUGUCCCCUACCAAGCUGAACGACCUGUGCUUCACAAACGUGUACGCCGACAGCUUCGUGAUCCGGGGAAACGAAGUGAGCCAGAUUGCCCCUGGACAGACAGGCAACAUCGCCGACUACAACUACAAGCUGCCCGACGACUUCACCGGCUGUGUGAUUGCCUGGAACAGCAACAAGCUGGACUCCAAAGUCGGCGGCAACUACAAUUACAGGUACCGGCUGUUCCGGAAGUCCAAUCUGAAGCCCUUCGAGCGGGACAUCUCCACCGAGAUCUAUCAGGCCGGCAACAAGCCUUGUAACGGCGUGGCCGGCGUGAACUGCUACUUCCCACUGCAGUCCUACGGCUUUAGGCCCACAUAUGGCGUGGGCCAUCAGCCCUACAGAGUGGUGGUGCUGAGCUUCGAACUGCUGCAUGCCCCUGCCACAGUGUGCGGCCCUAAGAAAAGCACCAAUCUCGUGAAGAACAAAUGCGUGAACUUCAACUUCAACGGCCUGACCGGCACCGGCGUGCUGACAGAGAGCAACAAGAAGUUCCUGCCAUUCCAGCAGUUUGGCCGGGAUAUCGCCGAUACCACAGACGCCGUUAGAGAUCCCCAGACACUGGAAAUCCUGGACAUCACCCCUUGCAGCUUCGGCGGAGUGUCUGUGAUCACCCCUGGCACCAACACCAGCAAUCAGGUGGCAGUGCUGUACCAGGGCGUGAACUGUACCGAAGUGCCCGUGGCCAUUCACGCCGAUCAGCUGACACCUACAUGGCGGGUGUACUCCACCGGCAGCAAUGUGUUUCAGACCAGAGCCGGCUGUCUGAUCGGAGCCGAGUACGUGAACAAUAGCUACGAGUGCGACAUCCCCAUCGGCGCUGGAAUCUGCGCCAGCUACCAGACACAGACAAAGAGCCACCGGAGAGCCAGAAGCGUGGCCAGCCAGAGCAUCAUUGCCUACACAAUGUCUCUGGGCGCCGAGAACAGCGUGGCCUACUCCAACAACUCUAUCGCUAUCCCCACCAACUUCACCAUCAGCGUGACCACAGAGAUCCUGCCUGUGUCCAUGACCAAGACCAGCGUGGACUGCACCAUGUACAUCUGCGGCGAUUCCACCGAGUGCUCCAACCUGCUGCUGCAGUACGGCAGCUUCUGCACCCAGCUGAAGAGAGCCCUGACAGGGAUCGCCGUGGAACAGGACAAGAACACCCAAGAGGUGUUCGCCCAAGUGAAGCAGAUCUACAAGACCCCUCCUAUCAAGUACUUCGGCGGCUUCAAUUUCAGCCAGAUUCUGCCCGAUCCUAGCAAGCCCAGCAAGCGGAGCUUCAUCGAGGACCUGCUGUUCAACAAAGUGACACUGGCCGACGCCGGCUUCAUCAAGCAGUAUGGCGAUUGUCUGGGCGACAUUGCCGCCAGGGAUCUGAUUUGCGCCCAGAAGUUUAACGGACUGACAGUGCUGCCUCCUCUGCUGACCGAUGAGAUGAUCGCCCAGUACACAUCUGCCCUGCUGGCCGGCACAAUCACAAGCGGCUGGACAUUUGGAGCAGGCGCCGCUCUGCAGAUCCCCUUUGCUAUGCAGAUGGCCUACCGGUUCAACGGCAUCGGAGUGACCCAGAAUGUGCUGUACGAGAACCAGAAGCUGAUCGCCAACCAGUUCAACAGCGCCAUCGGCAAGAUCCAGGACAGCCUGAGCAGCACAGCAAGCGCCCUGGGAAAGCUGCAGGACGUGGUCAACCACAAUGCCCAGGCACUGAACACCCUGGUCAAGCAGCUGUCCUCCAAGUUCGGCGCCAUCAGCUCUGUGCUGAACGAUAUCCUGAGCAGACUGGACCCUCCUGAGGCCGAGGUGCAGAUCGACAGACUGAUCACAGGCAGACUGCAGAGCCUCCAGACAUACGUGACCCAGCAGCUGAUCAGAGCCGCCGAGAUUAGAGCCUCUGCCAAUCUGGCCGCCACCAAGAUGUCUGAGUGUGUGCUGGGCCAGAGCAAGAGAGUGGACUUUUGCGGCAAGGGCUACCACCUGAUGAGCUUCCCUCAGUCUGCCCCUCACGGCGUGGUGUUUCUGCACGUGACAUAUGUGCCCGCUCAAGAGAAGAAUUUCACCACCGCUCCAGCCAUCUGCCACGACGGCAAAGCCCACUUUCCUAGAGAAGGCGUGUUCGUGUCCAACGGCACCCAUUGGUUCGUGACACAGCGGAACUUCUACGAGCCCCAGAUCAUCACCACCGACAACACCUUCGUGUCUGGCAACUGCGACGUCGUGAUCGGCAUUGUGAACAAUACCGUGUACGACCCUCUGCAGCCCGAGCUGGACAGCUUCAAAGAGGAACUGGACAAGUACUUUAAGAACCACACAAGCCCCGACGUGGACCUGGGCGAUAUCAGCGGAAUCAAUGCCAGCGUCGUGAACAUCCAGAAAGAGAUCGACCGGCUGAACGAGGUGGCCAAGAAUCUGAACGAGAGCCUGAUCGACCUGCAAGAACUGGGGAAGUACGAGCAGUACAUCAAGUGGCCCUGGUACAUCUGGCUGGGCUUUAUCGCCGGACUGAUUGCCAUCGUGAUGGUCACAAUCAUGCUGUGUUGCAUGACCAGCUGCUGUAGCUGCCUGAAGGGCUGUUGUAGCUGUGGCAGCUGCUGCAAGUUCGACGAGGACGAUUCUGAGCCCGUGCUGAAGGGCGUGAAACUGCACUACACAUGAUGA 76DNA sequence encoding aATGTTCGTGTTCCTGGTGCTGCTGCCTCTGGTGTCCAGCCAGTGTGTGAACCTGATCACCAGAACASARS-CoV-2 S protein from anCAGAGCTACACCAACAGCTTTACCAGAGGCGTGTACTACCCCGACAAGGTGTTCAGATCCAGCGTGOmicron BA.4/BA.5 variantCTGCACTCTACCCAGGACCTGTTCCTGCCTTTCTTCAGCAACGTGACCTGGTTCCACGCCATCTCC(with proline residues atGGCACCAATGGCACCAAGAGATTCGACAACCCCGTGCTGCCCTTCAACGACGGGGTGTACTTTGCCpositions corresponding toAGCACCGAGAAGTCCAACATCATCAGAGGCTGGATCTTCGGCACCACACTGGACAGCAAGACCCAGK986P and V987P of SEQ ID NO:AGCCTGCTGATCGTGAACAACGCCACCAACGTGGTCATCAAAGTGTGCGAGTTCCAGTTCTGCAAC 1)GACCCCTTCCTGGACGTGTACTACCACAAGAACAACAAGAGCTGGATGGAAAGCGAGTTCCGGGTGTACAGCAGCGCCAACAACTGCACCTTCGAGTACGTGTCCCAGCCTTTCCTGATGGACCTGGAAGGCAAGCAGGGCAACTTCAAGAACCTGCGCGAGTTCGTGTTTAAGAACATCGACGGCTACTTCAAGATCTACAGCAAGCACACCCCTATCAACCTCGGCCGGGATCTGCCTCAGGGCTTCTCTGCTCTGGAACCCCTGGTGGATCTGCCCATCGGCATCAACATCACCCGGTTTCAGACACTGCTGGCCCTGCACAGAAGCTACCTGACACCTGGCGATAGCAGCAGCGGATGGACAGCTGGTGCCGCCGCTTACTATGTGGGCTACCTGCAGCCTAGAACCTTCCTGCTGAAGTACAACGAGAACGGCACCATCACCGACGCCGTGGATTGTGCTCTGGATCCTCTGAGCGAGACAAAGTGCACCCTGAAGTCCTTCACCGTGGAAAAGGGCATCTACCAGACCAGCAACTTCCGGGTGCAGCCCACCGAATCCATCGTGCGGTTCCCCAATATCACCAATCTGTGCCCCTTCGACGAGGTGTTCAATGCCACCAGATTCGCCTCTGTGTACGCCTGGAACCGGAAGCGGATCAGCAATTGCGTGGCCGACTACTCCGTGCTGTACAACTTCGCCCCCTTCTTCGCCTTCAAGTGCTACGGCGTGTCCCCTACCAAGCTGAACGACCTGTGCTTCACAAACGTGTACGCCGACAGCTTCGTGATCCGGGGAAACGAAGTGAGCCAGATTGCCCCTGGACAGACAGGCAACATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGTGTGATTGCCTGGAACAGCAACAAGCTGGACTCCAAAGTCGGCGGCAACTACAATTACAGGTACCGGCTGTTCCGGAAGTCCAATCTGAAGCCCTTCGAGCGGGACATCTCCACCGAGATCTATCAGGCCGGCAACAAGCCTTGTAACGGCGTGGCCGGCGTGAACTGCTACTTCCCACTGCAGTCCTACGGCTTTAGGCCCACATATGGCGTGGGCCATCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAACTGCTGCATGCCCCTGCCACAGTGTGCGGCCCTAAGAAAAGCACCAATCTCGTGAAGAACAAATGCGTGAACTTCAACTTCAACGGCCTGACCGGCACCGGCGTGCTGACAGAGAGCAACAAGAAGTTCCTGCCATTCCAGCAGTTTGGCCGGGATATCGCCGATACCACAGACGCCGTTAGAGATCCCCAGACACTGGAAATCCTGGACATCACCCCTTGCAGCTTCGGCGGAGTGTCTGTGATCACCCCTGGCACCAACACCAGCAATCAGGTGGCAGTGCTGTACCAGGGCGTGAACTGTACCGAAGTGCCCGTGGCCATTCACGCCGATCAGCTGACACCTACATGGCGGGTGTACTCCACCGGCAGCAATGTGTTTCAGACCAGAGCCGGCTGTCTGATCGGAGCCGAGTACGTGAACAATAGCTACGAGTGCGACATCCCCATCGGCGCTGGAATCTGCGCCAGCTACCAGACACAGACAAAGAGCCACCGGAGAGCCAGAAGCGTGGCCAGCCAGAGCATCATTGCCTACACAATGTCTCTGGGCGCCGAGAACAGCGTGGCCTACTCCAACAACTCTATCGCTATCCCCACCAACTTCACCATCAGCGTGACCACAGAGATCCTGCCTGTGTCCATGACCAAGACCAGCGTGGACTGCACCATGTACATCTGCGGCGATTCCACCGAGTGCTCCAACCTGCTGCTGCAGTACGGCAGCTTCTGCACCCAGCTGAAGAGAGCCCTGACAGGGATCGCCGTGGAACAGGACAAGAACACCCAAGAGGTGTTCGCCCAAGTGAAGCAGATCTACAAGACCCCTCCTATCAAGTACTTCGGCGGCTTCAATTTCAGCCAGATTCTGCCCGATCCTAGCAAGCCCAGCAAGCGGAGCTTCATCGAGGACCTGCTGTTCAACAAAGTGACACTGGCCGACGCCGGCTTCATCAAGCAGTATGGCGATTGTCTGGGCGACATTGCCGCCAGGGATCTGATTTGCGCCCAGAAGTTTAACGGACTGACAGTGCTGCCTCCTCTGCTGACCGATGAGATGATCGCCCAGTACACATCTGCCCTGCTGGCCGGCACAATCACAAGCGGCTGGACATTTGGAGCAGGCGCCGCTCTGCAGATCCCCTTTGCTATGCAGATGGCCTACCGGTTCAACGGCATCGGAGTGACCCAGAATGTGCTGTACGAGAACCAGAAGCTGATCGCCAACCAGTTCAACAGCGCCATCGGCAAGATCCAGGACAGCCTGAGCAGCACAGCAAGCGCCCTGGGAAAGCTGCAGGACGTGGTCAACCACAATGCCCAGGCACTGAACACCCTGGTCAAGCAGCTGTCCTCCAAGTTCGGCGCCATCAGCTCTGTGCTGAACGATATCCTGAGCAGACTGGACCCTCCTGAGGCCGAGGTGCAGATCGACAGACTGATCACAGGCAGACTGCAGAGCCTCCAGACATACGTGACCCAGCAGCTGATCAGAGCCGCCGAGATTAGAGCCTCTGCCAATCTGGCCGCCACCAAGATGTCTGAGTGTGTGCTGGGCCAGAGCAAGAGAGTGGACTTTTGCGGCAAGGGCTACCACCTGATGAGCTTCCCTCAGTCTGCCCCTCACGGCGTGGTGTTTCTGCACGTGACATATGTGCCCGCTCAAGAGAAGAATTTCACCACCGCTCCAGCCATCTGCCACGACGGCAAAGCCCACTTTCCTAGAGAAGGCGTGTTCGTGTCCAACGGCACCCATTGGTTCGTGACACAGCGGAACTTCTACGAGCCCCAGATCATCACCACCGACAACACCTTCGTGTCTGGCAACTGCGACGTCGTGATCGGCATTGTGAACAATACCGTGTACGACCCTCTGCAGCCCGAGCTGGACAGCTTCAAAGAGGAACTGGACAAGTACTTTAAGAACCACACAAGCCCCGACGTGGACCTGGGCGATATCAGCGGAATCAATGCCAGCGTCGTGAACATCCAGAAAGAGATCGACCGGCTGAACGAGGTGGCCAAGAATCTGAACGAGAGCCTGATCGACCTGCAAGAACTGGGGAAGTACGAGCAGTACATCAAGTGGCCCTGGTACATCTGGCTGGGCTTTATCGCCGGACTGATTGCCATCGTGATGGTCACAATCATGCTGTGTTGCATGACCAGCTGCTGTAGCTGCCTGAAGGGCTGTTGTAGCTGTGGCAGCTGCTGCAAGTTCGACGAGGACGATTCTGAGCCCGTGCTGAAGGGCGTGAAACTGCACTACACATGATGA

TABLE 11Sequence of one embodiment of Omicron BA.4/BA.5-specific RNA vaccine SEQID NO. Brief Description Sequence 77 Amino acid sequence of RNA-MFVFLVLLPLVSSQCVNLITRTQSYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAISencoded SARS-CoV-2 S proteinGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNfrom an Omicron BA.4/BA.5DPFLDVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIvariant (with PRO mutationsYSKHTPINLGRDLPQGFSALEPLVDLPIGINITREQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYat positions corresponding toLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNERVQPTESIVRFPNITNLK986P and V987P of SEQ ID NO:CPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFV1; i.e., PRO mutations atIRGNEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVGGNYNYRYRLFRKSNLKPFERDpositions 981 and 982 of SEQISTEIYQAGNKPCNGVAGVNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLID NO: 77)VKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEYVNNSYECDIPIGAGICASYQTQTKSHRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLKRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKYFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKENGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRENGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNHNAQALNTLVKQLSSKFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNETTAPAICHDGKAHFPREGVEVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKEDEDDSEPVLKGVKLHYT 78 RNA sequence encoding a SARS-AUGUUCGUGUUCCUGGUGCUGCUGCCUCUGGUGUCCAGCCAGUGUGUGAACCUGAUCACCAGAACACoV-2 S protein from anCAGUCAUACACCAACAGCUUUACCAGAGGCGUGUACUACCCCGACAAGGUGUUCAGAUCCAGCGUGOmicron BA.4/BA.5 variantCUGCACUCUACCCAGGACCUGUUCCUGCCUUUCUUCAGCAACGUGACCUGGUUCCACGCCAUCUCCGGCACCAAUGGCACCAAGAGAUUCGACAACCCCGUGCUGCCCUUCAACGACGGGGUGUACUUUGCCAGCACCGAGAAGUCCAACAUCAUCAGAGGCUGGAUCUUCGGCACCACACUGGACAGCAAGACCCAGAGCCUGCUGAUCGUGAACAACGCCACCAACGUGGUCAUCAAAGUGUGCGAGUUCCAGUUCUGCAACGACCCCUUCCUGGACGUCUACUACCACAAGAACAACAAGAGCUGGAUGGAAAGCGAGUUCCGGGUGUACAGCAGCGCCAACAACUGCACCUUCGAGUACGUGUCCCAGCCUUUCCUGAUGGACCUGGAAGGCAAGCAGGGCAACUUCAAGAACCUGCGCGAGUUCGUGUUUAAGAACAUCGACGGCUACUUCAAGAUCUACAGCAAGCACACCCCUAUCAACCUCGGCCGGGAUCUGCCUCAGGGCUUCUCUGCUCUGGAACCCCUGGUGGAUCUGCCCAUCGGCAUCAACAUCACCCGGUUUCAGACACUGCUGGCCCUGCACAGAAGCUACCUGACACCUGGCGAUAGCAGCAGCGGAUGGACAGCUGGUGCCGCCGCUUACUAUGUGGGCUACCUGCAGCCUAGAACCUUCCUGCUGAAGUACAACGAGAACGGCACCAUCACCGACGCCGUGGAUUGUGCUCUGGAUCCUCUGAGCGAGACAAAGUGCACCCUGAAGUCCUUCACCGUGGAAAAGGGCAUCUACCAGACCAGCAACUUCCGGGUGCAGCCCACCGAAUCCAUCGUGCGGUUCCCCAAUAUCACCAAUCUGUGCCCCUUCGACGAGGUGUUCAAUGCCACCAGAUUCGCCUCUGUGUACGCCUGGAACCGGAAGCGGAUCAGCAAUUGCGUGGCCGACUACUCCGUGCUGUACAACUUCGCCCCCUUCUUCGCAUUCAAGUGCUACGGCGUGUCCCCUACCAAGCUGAACGACCUGUGCUUCACAAACGUGUACGCCGACAGCUUCGUGAUCCGGGGAAACGAAGUGCGGCAGAUUGCCCCUGGACAGACAGGCAACAUCGCCGACUACAACUACAAGCUGCCCGACGACUUCACCGGCUGUGUGAUUGCCUGGAACAGCAACAAGCUGGACUCCAAAGUCGGCGGCAACUACAAUUACAGGUACCGGCUGUUCCGGAAGUCCAAUCUGAAGCCCUUCGAGCGGGACAUCUCCACCGAGAUCUAUCAGGCCGGCAACAAGCCUUGUAACGGCGUGGCAGGCGUGAACUGCUACUUCCCACUGCAGUCCUACGGCUUUAGGCCCACAUACGGCGUGGGCCACCAGCCCUACAGAGUGGUGGUGCUGAGCUUCGAACUGCUGCAUGCCCCUGCCACAGUGUGCGGCCCUAAGAAAAGCACCAAUCUCGUGAAGAACAAAUGCGUGAACUUCAACUUCAACGGCCUGACCGGCACCGGCGUGCUGACAGAGAGCAACAAGAAGUUCCUGCCAUUCCAGCAGUUUGGCCGGGAUAUCGCCGAUACCACAGACGCCGUUAGAGAUCCCCAGACACUGGAAAUCCUGGACAUCACCCCUUGCAGCUUCGGCGGAGUGUCUGUGAUCACCCCUGGCACCAACACCAGCAAUCAGGUGGCAGUGCUGUACCAGGGCGUGAACUGUACCGAAGUGCCCGUGGCCAUUCACGCCGAUCAGCUGACACCUACAUGGCGGGUGUACUCCACCGGCAGCAAUGUGUUUCAGACCAGAGCCGGCUGUCUGAUCGGAGCCGAGUACGUGAACAAUAGCUACGAGUGCGACAUCCCCAUCGGCGCUGGAAUCUGCGCCAGCUACCAGACACAGACAAAGAGCCACCGGAGAGCCAGAAGCGUGGCCAGCCAGAGCAUCAUUGCCUACACAAUGUCUCUGGGCGCCGAGAACAGCGUGGCCUACUCCAACAACUCUAUCGCUAUCCCCACCAACUUCACCAUCAGCGUGACCACAGAGAUCCUGCCUGUGUCCAUGACCAAGACCAGCGUGGACUGCACCAUGUACAUCUGCGGCGAUUCCACCGAGUGCUCCAACCUGCUGCUGCAGUACGGCAGCUUCUGCACCCAGCUGAAAAGAGCCCUGACAGGGAUCGCCGUGGAACAGGACAAGAACACCCAAGAGGUGUUCGCCCAAGUGAAGCAGAUCUACAAGACCCCUCCUAUCAAGUACUUCGGCGGCUUCAAUUUCAGCCAGAUUCUGCCCGAUCCUAGCAAGCCCAGCAAGCGGAGCUUCAUCGAGGACCUGCUGUUCAACAAAGUGACACUGGCCGACGCCGGCUUCAUCAAGCAGUAUGGCGAUUGUCUGGGCGACAUUGCCGCCAGGGAUCUGAUUUGCGCCCAGAAGUUUAACGGACUGACAGUGCUGCCUCCUCUGCUGACCGAUGAGAUGAUCGCCCAGUACACAUCUGCCCUGCUGGCCGGCACAAUCACAAGCGGCUGGACAUUUGGAGCAGGCGCCGCUCUGCAGAUCCCCUUUGCUAUGCAGAUGGCCUACCGGUUCAACGGCAUCGGAGUGACCCAGAAUGUGCUGUACGAGAACCAGAAGCUGAUCGCCAACCAGUUCAACAGCGCCAUCGGCAAGAUCCAGGACAGCCUGAGCAGCACAGCAAGCGCCCUGGGAAAGCUGCAGGACGUGGUCAACCACAAUGCCCAGGCACUGAACACCCUGGUCAAGCAGCUGUCCUCCAAGUUCGGCGCCAUCAGCUCUGUGCUGAACGAUAUCCUGAGCAGACUGGACCCUCCUGAGGCCGAGGUGCAGAUCGACAGACUGAUCACAGGCAGACUGCAGAGCCUCCAGACAUACGUGACCCAGCAGCUGAUCAGAGCCGCCGAGAUUAGAGCCUCUGCCAAUCUGGCCGCCACCAAGAUGUCUGAGUGUGUGCUGGGCCAGAGCAAGAGAGUGGACUUUUGCGGCAAGGGCUACCACCUGAUGAGCUUCCCUCAGUCUGCCCCUCACGGCGUGGUGUUUCUGCACGUGACAUAUGUGCCCGCUCAAGAGAAGAAUUUCACCACCGCUCCAGCCAUCUGCCACGACGGCAAAGCCCACUUUCCUAGAGAAGGCGUGUUCGUGUCCAACGGCACCCAUUGGUUCGUGACACAGCGGAACUUCUACGAGCCCCAGAUCAUCACCACCGACAACACCUUCGUGUCUGGCAACUGCGACGUCGUGAUCGGCAUUGUGAACAAUACCGUGUACGACCCUCUGCAGCCCGAGCUGGACAGCUUCAAAGAGGAACUGGACAAGUACUUUAAGAACCACACAAGCCCCGACGUGGACCUGGGCGAUAUCAGCGGAAUCAAUGCCAGCGUCGUGAACAUCCAGAAAGAGAUCGACCGGCUGAACGAGGUGGCCAAGAAUCUGAACGAGAGCCUGAUCGACCUGCAAGAACUGGGGAAGUACGAGCAGUACAUCAAGUGGCCCUGGUACAUCUGGCUGGGCUUUAUCGCCGGACUGAUUGCCAUCGUGAUGGUCACAAUCAUGCUGUGUUGCAUGACCAGCUGCUGUAGCUGCCUGAAGGGCUGUUGUAGCUGUGGCAGCUGCUGCAAGUUCGACGAGGACGAUUCUGAGCCCGUGCUGAAGGGCGUGAAACUGCACUACACAUGAUGA 79DNA sequence encoding a SARS-ATGTTCGTGTTCCTGGTGCTGCTGCCTCTGGTGTCCAGCCAGTGTGTGAACCTGATCACCAGAACACoV-2 S protein from anCAGTCATACACCAACAGCTTTACCAGAGGCGTGTACTACCCCGACAAGGTGTTCAGATCCAGCGTGOmicron BA.4/BA.5 variantCTGCACTCTACCCAGGACCTGTTCCTGCCTTTCTTCAGCAACGTGACCTGGTTCCACGCCATCTCCGGCACCAATGGCACCAAGAGATTCGACAACCCCGTGCTGCCCTTCAACGACGGGGTGTACTTTGCCAGCACCGAGAAGTCCAACATCATCAGAGGCTGGATCTTCGGCACCACACTGGACAGCAAGACCCAGAGCCTGCTGATCGTGAACAACGCCACCAACGTGGTCATCAAAGTGTGCGAGTTCCAGTTCTGCAACGACCCCTTCCTGGACGTCTACTACCACAAGAACAACAAGAGCTGGATGGAAAGCGAGTTCCGGGTGTACAGCAGCGCCAACAACTGCACCTTCGAGTACGTGTCCCAGCCTTTCCTGATGGACCTGGAAGGCAAGCAGGGCAACTTCAAGAACCTGCGCGAGTTCGTGTTTAAGAACATCGACGGCTACTTCAAGATCTACAGCAAGCACACCCCTATCAACCTCGGCCGGGATCTGCCTCAGGGCTTCTCTGCTCTGGAACCCCTGGTGGATCTGCCCATCGGCATCAACATCACCCGGTTTCAGACACTGCTGGCCCTGCACAGAAGCTACCTGACACCTGGCGATAGCAGCAGCGGATGGACAGCTGGTGCCGCCGCTTACTATGTGGGCTACCTGCAGCCTAGAACCTTCCTGCTGAAGTACAACGAGAACGGCACCATCACCGACGCCGTGGATTGTGCTCTGGATCCTCTGAGCGAGACAAAGTGCACCCTGAAGTCCTTCACCGTGGAAAAGGGCATCTACCAGACCAGCAACTTCCGGGTGCAGCCCACCGAATCCATCGTGCGGTTCCCCAATATCACCAATCTGTGCCCCTTCGACGAGGTGTTCAATGCCACCAGATTCGCCTCTGTGTACGCCTGGAACCGGAAGCGGATCAGCAATTGCGTGGCCGACTACTCCGTGCTGTACAACTTCGCCCCCTTCTTCGCATTCAAGTGCTACGGCGTGTCCCCTACCAAGCTGAACGACCTGTGCTTCACAAACGTGTACGCCGACAGCTTCGTGATCCGGGGAAACGAAGTGCGGCAGATTGCCCCTGGACAGACAGGCAACATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGTGTGATTGCCTGGAACAGCAACAAGCTGGACTCCAAAGTCGGCGGCAACTACAATTACAGGTACCGGCTGTTCCGGAAGTCCAATCTGAAGCCCTTCGAGCGGGACATCTCCACCGAGATCTATCAGGCCGGCAACAAGCCTTGTAACGGCGTGGCAGGCGTGAACTGCTACTTCCCACTGCAGTCCTACGGCTTTAGGCCCACATACGGCGTGGGCCACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAACTGCTGCATGCCCCTGCCACAGTGTGCGGCCCTAAGAAAAGCACCAATCTCGTGAAGAACAAATGCGTGAACTTCAACTTCAACGGCCTGACCGGCACCGGCGTGCTGACAGAGAGCAACAAGAAGTTCCTGCCATTCCAGCAGTTTGGCCGGGATATCGCCGATACCACAGACGCCGTTAGAGATCCCCAGACACTGGAAATCCTGGACATCACCCCTTGCAGCTTCGGCGGAGTGTCTGTGATCACCCCTGGCACCAACACCAGCAATCAGGTGGCAGTGCTGTACCAGGGCGTGAACTGTACCGAAGTGCCCGTGGCCATTCACGCCGATCAGCTGACACCTACATGGCGGGTGTACTCCACCGGCAGCAATGTGTTTCAGACCAGAGCCGGCTGTCTGATCGGAGCCGAGTACGTGAACAATAGCTACGAGTGCGACATCCCCATCGGCGCTGGAATCTGCGCCAGCTACCAGACACAGACAAAGAGCCACCGGAGAGCCAGAAGCGTGGCCAGCCAGAGCATCATTGCCTACACAATGTCTCTGGGCGCCGAGAACAGCGTGGCCTACTCCAACAACTCTATCGCTATCCCCACCAACTTCACCATCAGCGTGACCACAGAGATCCTGCCTGTGTCCATGACCAAGACCAGCGTGGACTGCACCATGTACATCTGCGGCGATTCCACCGAGTGCTCCAACCTGCTGCTGCAGTACGGCAGCTTCTGCACCCAGCTGAAAAGAGCCCTGACAGGGATCGCCGTGGAACAGGACAAGAACACCCAAGAGGTGTTCGCCCAAGTGAAGCAGATCTACAAGACCCCTCCTATCAAGTACTTCGGCGGCTTCAATTTCAGCCAGATTCTGCCCGATCCTAGCAAGCCCAGCAAGCGGAGCTTCATCGAGGACCTGCTGTTCAACAAAGTGACACTGGCCGACGCCGGCTTCATCAAGCAGTATGGCGATTGTCTGGGCGACATTGCCGCCAGGGATCTGATTTGCGCCCAGAAGTTTAACGGACTGACAGTGCTGCCTCCTCTGCTGACCGATGAGATGATCGCCCAGTACACATCTGCCCTGCTGGCCGGCACAATCACAAGCGGCTGGACATTTGGAGCAGGCGCCGCTCTGCAGATCCCCTTTGCTATGCAGATGGCCTACCGGTTCAACGGCATCGGAGTGACCCAGAATGTGCTGTACGAGAACCAGAAGCTGATCGCCAACCAGTTCAACAGCGCCATCGGCAAGATCCAGGACAGCCTGAGCAGCACAGCAAGCGCCCTGGGAAAGCTGCAGGACGTGGTCAACCACAATGCCCAGGCACTGAACACCCTGGTCAAGCAGCTGTCCTCCAAGTTCGGCGCCATCAGCTCTGTGCTGAACGATATCCTGAGCAGACTGGACCCTCCTGAGGCCGAGGTGCAGATCGACAGACTGATCACAGGCAGACTGCAGAGCCTCCAGACATACGTGACCCAGCAGCTGATCAGAGCCGCCGAGATTAGAGCCTCTGCCAATCTGGCCGCCACCAAGATGTCTGAGTGTGTGCTGGGCCAGAGCAAGAGAGTGGACTTTTGCGGCAAGGGCTACCACCTGATGAGCTTCCCTCAGTCTGCCCCTCACGGCGTGGTGTTTCTGCACGTGACATATGTGCCCGCTCAAGAGAAGAATTTCACCACCGCTCCAGCCATCTGCCACGACGGCAAAGCCCACTTTCCTAGAGAAGGCGTGTTCGTGTCCAACGGCACCCATTGGTTCGTGACACAGCGGAACTTCTACGAGCCCCAGATCATCACCACCGACAACACCTTCGTGTCTGGCAACTGCGACGTCGTGATCGGCATTGTGAACAATACCGTGTACGACCCTCTGCAGCCCGAGCTGGACAGCTTCAAAGAGGAACTGGACAAGTACITTAAGAACCACACAAGCCCCGACGTGGACCTGGGCGATATCAGCGGAATCAATGCCAGCGTCGTGAACATCCAGAAAGAGATCGACCGGCTGAACGAGGTGGCCAAGAATCTGAACGAGAGCCTGATCGACCTGCAAGAACTGGGGAAGTACGAGCAGTACATCAAGTGGCCCTGGTACATCTGGCTGGGCTTTATCGCCGGACTGATTGCCATCGTGATGGTCACAATCATGCTGTGTTGCATGACCAGCTGCTGTAGCTGCCTGAAGGGCTGTTGTAGCTGTGGCAGCTGCTGCAAGTTCGACGAGGACGATTCTGAGCCCGTGCTGAAGGGCGTGAAACTGCACTACACATGATGA

TABLE 12Sequence of one embodiment of an exemplary Omicron BA.2.75-specific RNA vaccineSEQ ID NO. Brief Description Sequence 80 Amino acid sequence of RNA-MFVFLVLLPLVSSQCVNLITRTQSYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHencoded SARS-CoV-2 S proteinVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIEGTTLDSKTQSLLIVNNATNVVIKVCEFQFfrom an Omicron BA.2.75CNDPFLDVYYHENNKSRMESELRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYEvariant (with PRO mutations atKIYSKHTPVNLGRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSSWTAGAAAYYVpositions corresponding toGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFRVEKFIYQTSNFRVQPTESIVRFPNITpositions K986P and V987P ofNLCPFHEVFNATRFASVYAWNRKRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSSEQ ID NO: 1; i.e., PROFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVSGNYNYLYRLFRKSKLKPFEmutations at positions 983 andRDISTEIYQAGNKPCNGVAGENCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKST984 of SEQ ID NO: 80)NLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEYVNNSYECDIPIGAGICASYQTQTKSHRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNETISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLKRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKYFGGFNFSQILPDPSKPSKRSFIEDLLENKVTLADAGFIKQYGDCLGDIAARDLICAQKENGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNHNAQALNTLVKQLSSKFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVEVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT 81 RNA sequence encoding a SARS-auguucguguuccuggugcugcugccucugguguccagccagugugugaaccugaucaccagaacaCoV-2 S protein from ancagucauacaccaacagcuuuaccagaggcguguacuaccccgacaagguguucagauccagcgugOmicron BA.2.75 variantcugcacucuacccaggaccuguuccugccuuucuucagcaacgugaccugguuccacgccauccacguguccggcaccaauggcaccaagagauucgacaaccccgugcugcccuucaacgacgggguguacuuugccagcaccgagaaguccaacaucaucagaggcuggaucuucggcaccacacuggacagcaagacccagagccugcugaucgugaacaacgccaccaacguggucaucaaagugugcgaguuccaguucugcaacgaccccuuccuggacgucuacuaccacgagaacaacaagagcaggauggaaagcgagcuccggguguacagcagcgccaacaacugcaccuucgaguacgugucccagccuuuccugauggaccuggaaggcaagcagggcaacuucaagaaccugcgcgaguucguguuuaagaacaucgacggcuacuucaagaucuacagcaagcacaccccugugaaccucggccgggaucugccucagggcuucucugcucuggaaccccugguggaucugcccaucggcaucaacaucacccgguuucagacacugcuggcccugcacagaagcuaccugacaccuggcgauagcagcagcagcuggacagcuggugccgccgcuuacuaugugggcuaccugcagccuagaaccuuccugcugaaquacaacgagaacggcaccaucaccgacgccguggauugugcucuggauccucugagcgagacaaagugcacccugaaguccuucaccguggaaaagggcaucuaccagaccagcaacuuccgggugcagcccaccgaauccaucgugcgguuccccaauaucaccaaucugugccccuuccacgagguguucaaugccaccagauucgccucuguguacgccuggaaccggaagcggaucagcaauugcguggccgacuacuccgugcuguacaacuucgcccccuucuucgcauucaagugcuacggcguguccccuaccaagcugaacgaccugugcuucacaaacguguacgccgacagcuucgugauccggggaaacgaagugucacagauugccccuggacagacaggcaacaucgccgacuacaacuacaagcugcccgacgacuucaccggcugugugauugccuggaacagcaacaagcuggacuccaaagucagcggcaacuacaauuaccuguaccggcuguuccggaaguccaagcugaagcccuucgagcgggacaucuccaccgagaucuaucaggccggcaacaagccuuguaacggcguggcaggcuucaacugcuacuucccacugcaguccuacggcuuuaggcccacauacggcgugggccaccagcccuacagagugguggugcugagcuucgaacugcugcaugccccugccacagugugcggcccuaagaaaagcaccaaucucgugaagaacaaaugcgugaacuucaacuucaacggccugaccggcaccggcgugcugacagagagcaacaagaaguuccugccauuccagcaguuuggccgggauaucgccgauaccacagacgccguuagagauccccagacacuggaaauccuggacaucaccccuugcagcuucggcggagugucugugaucaccccuggcaccaacaccagcaaucagguggcagugcuguaccagggcgugaacuguaccgaagugcccguggccauucacgccgaucagcugacaccuacauggcggguguacuccaccggcagcaauguguuucagaccagagccggcugucugaucggagccgaguacgugaacaauagcuacgagugcgacauccccaucggcgcuggaaucugcgccagcuaccagacacagacaaagagccaccggagagccagaagcguggccagccagagcaucauugccuacacaaugucucugggcgccgagaacagcguggccuacuccaacaacucuaucgcuauccccaccaacuucaccaucagcgugaccacagagauccugccuguguccaugaccaagaccagcguggacugcaccauguacaucugcggcgauuccaccgagugcuccaaccugcugcugcaguacggcagcuucugcacccagcugaaaagagcccugacagggaucgccguggaacaggacaagaacacccaagagguguucgcccaagugaagcagaucuacaagaccccuccuaucaaguacuucggcggcuucaauuucagccagauucugcccgauccuagcaagcccagcaagcggagcuucaucgaggaccugcuguucaacaaagugacacuggccgacgccggcuucaucaagcaguauggcgauugucugggcgacauugccgccagggaucugauuugcgcccagaaguuuaacggacugacagugcugccuccucugcugaccgaugagaugaucgcccaguacacaucugcccugcuggccggcacaaucacaagcggcuggacauuuggagcaggcgccgcucugcagauccccuuugcuaugcagauggccuaccgguucaacggcaucggagugacccagaaugugcuguacgagaaccagaagcugaucgccaaccaguucaacagcgccaucggcaagauccaggacagccugagcagcacagcaagcgcccugggaaagcugcaggacguggucaaccacaaugcccaggcacugaacacccuggucaagcagcuguccuccaaguucggcgccaucagcucugugcugaacgauauccugagcagacuggacccuccugaggccgaggugcagaucgacagacugaucacaggcagacugcagagccuccagacauacgugacccagcagcugaucagagccgccgagauuagagccucugccaaucuggccgccaccaagaugucugagugugugcugggccagagcaagagaguggacuuuugcggcaagggcuaccaccugaugagcuucccucagucugccccucacggcgugguguuucugcacgugacauaugugcccgcucaagagaagaauuucaccaccgcuccagccaucugccacgacggcaaagcccacuuuccuagagaaggcguguucguguccaacggcacccauugguucgugacacagcggaacuucuacgagccccagaucaucaccaccgacaacaccuucgugucuggcaacugcgacqucgugaucggcauugugaacaauaccguguacgacccucugcagcccgagcuggacagcuucaaagaggaacuggacaaguacuuuaagaaccacacaagccccgacguggaccugggcgauaucagcggaaucaaugccagcgucgugaacauccagaaagagaucgaccggcugaacgagguggccaagaaucugaacgagagccugaucgaccugcaagaacuggggaaguacgagcaguacaucaaguggcccugguacaucuggcugggcuuuaucgccggacugauugccaucgugauggucacaaucaugcuguguugcaugaccagcugcuguagcugccugaagggcuguuguagcuguggcagcugcugcaaguucgacgaggacgauucugagcccgugcugaagggcgugaaacugcacuacacaugauga 82DNA sequence encoding a SARS-atgttcgtgttcctggtgctgctgcctctggtgtccagccagtgtgtgaacctgatcaccagaacaCoV-2 S protein from ancagtcatacaccaacagctttaccagaggcgtgtactaccccgacaaggtgttcagatccagcgtgOmicron BA.2.75 variantctgcactgtacccaggacctgttcctgcctttcttcagcaacgtgacctggttccacgccatccacgtgtccggcaccaatggcaccaagagattcgacaaccccgtgctgcccttcaacgacggggtgtactttgccagcaccgagaagtccaacatcatcagaggctggatcttcggcaccacactggacagcaagacccagagcctgctgatcgtgaacaacgccaccaacgtggtcatcaaagtgtgcgagttccagttctgcaacgaccccttcctggacgtctactaccacgagaacaacaagagcaggatggaaagcgagctccgggtgtacagcagcgccaacaactgcaccttcgagtacgtgtcccagcctttcctgatggacctggaaggcaagcagggcaacttcaagaacctgcgcgagttcgtgtttaagaacatcgacggctacttcaagatctacagcaagcacacccctgtgaacctcggccgggatctgcctcagggcttctctgctctggaacccctggtggatctgcccatcggcatcaacatcacccggtttcagacactgctggccctgcacagaagctacctgacacctggcgatagcagcagcagctggacagctggtgccgccgcttactatgtgggctacctgcagcctagaaccttcctgctgaagtacaacgagaacggcaccatcaccgacgccgtggattgtgctctggatcctctgagcgagacaaagtgcaccctgaagtccttcaccgtggaaaagggcatctaccagaccagcaacttccgggtgcagcccaccgaatccatcgtgcggttccccaatatcaccaatctgtgccccttccacgaggtgttcaatgccaccagattcgcctctgtgtacgcctggaaccggaagcggatcagcaattgcgtggccgactactccgtgctgtacaacttcgcccccttcttcgcattcaagtgctacggcgtgtcccctaccaagctgaacgacctgtgcttcacaaacgtgtacgccgacagcttcgtgatccggggaaacgaagtgtcacagattgcccctggacagacaggcaacatcgccgactacaactacaagctgcccgacgacttcaccggctgtgtgattgcctggaacagcaacaagctggactccaaagtcagcggcaactacaattacctgtaccggctgttccggaagtccaagctgaagcccttcgagcgggacatctccaccgagatctatcaggccggcaacaagccttgtaacggcgtggcaggcttcaactgctacttcccactgcagtcctacggctttaggcccacatacggcgtgggccaccagccctacagagtggtggtgctgagcttcgaactgctgcatgcccctgccacagtgtgcggccctaagaaaagcaccaatctcgtgaagaacaaatgcgtgaacttcaacttcaacggcctgaccggcaccggcgtgctgacagagagcaacaagaagttcctgccattccagcagtttggccgggatatcgccgataccacagacgccgttagagatccccagacactggaaatcctggacatcaccccttgcagcttcggcggagtgtctgtgatcacccctggcaccaacaccagcaatcaggtggcagtgctgtaccagggcgtgaactgtaccgaagtgcccgtggccattcacgccgatcagctgacacctacatggcgggtgtactccaccggcagcaatgtgtttcagaccagagccggctgtctgatcggagccgagtacgtgaacaatagctacgagtgcgacatccccatcggcgctggaatctgcgccagctaccagacacagacaaagagccaccggagagccagaagcgtggccagccagagcatcattgcctacacaatgtctctgggcgccgagaacagcgtggcctactccaacaactctatcgctatccccaccaacttcaccatcagcgtgaccacagagatcctgcctgtgtccatgaccaagaccagcgtggactgcaccatgtacatctgcggcgattccaccgagtgctccaacctgctgctgcagtacggcagcttctgcacccagctgaaaagagccctgacagggatcgccgtggaacaggacaagaacacccaagaggtgttcgcccaagtgaagcagatctacaagacccctcctatcaagtacttcggcggcttcaatttcagccagattctgcccgatcctagcaagcccagcaagcggagcttcatcgaggacctgctgttcaacaaagtgacactggccgacgccggcttcatcaagcagtatggcgattgtctgggcgacattgccgccagggatctgatttgcgcccagaagtttaacggactgacagtgctgcctcctctgctgaccgatgagatgatcgcccagtacacatctgccctgctggccggcacaatcacaagcggctggacatttggagcaggcgccgctctgcagatcccctttgctatgcagatggcctaccggttcaacggcatcggagtgacccagaatgtgctgtacgagaaccagaagctgatcgccaaccagttcaacagcgccatcggcaagatccaggacagcctgagcagcacagcaagcgccctgggaaagctgcaggacgtggtcaaccacaatgcccaggcactgaacaccctggtcaagcagctgtcctccaagttcggcgccatcagctctgtgctgaacgatatcctgagcagactggaccctcctgaggccgaggtgcagatcgacagactgatcacaggcagactgcagagcctccagacatacgtgacccagcagctgatcagagccgccgagattagagcctctgccaatctggccgccaccaagatgtctgagtgtgtgctgggccagagcaagagagtggacttttgcggcaagggctaccacctgatgagcttccctcagtctgcccctcacggcgtggtgtttctgcacgtgacatatgtgcccgctcaagagaagaatttcaccaccgctccagccatctgccacgacggcaaagcccactttcctagagaaggcgtgttcgtgtccaacggcacccattggttcgtgacacagcggaacttctacgagccccagatcatcaccaccgacaacaccttcgtgtctggcaactgcgacgtcgtgatcggcattgtgaacaataccgtgtacgaccctctqcagcccgagctggacagcttcaaagaggaactggacaagtactttaagaaccacacaagccccgacgtggacctgggcgatatcagcggaatcaatgccagcgtcgtgaacatccagaaagagatcgaccggctgaacgaggtggccaagaatctgaacgagagcctgatcgacctgcaagaactggggaagtacgagcagtacatcaagtggccctggtacatctggctgggctttatcgccggactgattgccatcgtgatggtcacaatcatgctgtgttgcatgaccagctgctgtagctgcctgaagggctgttgtagctgtggcagctgctgcaagttcgacgaggacgattctgagcccgtgctgaagggcgtgaaactgcactacacatgatga 83Full length RNA constructagaauaaacuaguauucuucugguccccacagacucagagagaacccgccaccauguucguguuccencoding a SARS-Cov-2 Suggugcugcugccucugguguccagccagugugugaaccugaucaccagaacacagucauacaccaprotein from an OmicronacagcuuuaccagaggcguguacuaccccgacaagguguucagauccagcgugcugcacucuacccBA.2.75 variantaggaccuguuccugccuuucuucagcaacgugaccugguuccacgccauccacguguccggcaccaauggcaccaagagauucgacaaccccgugcugcccuucaacgacgggguguacuuugccagcaccgagaaguccaacaucaucagaggcuggaucuucggcaccacacuggacagcaagacccagagccugcugaucgugaacaacgccaccaacguggucaucaaagugugcgaguuccaguucugcaacgaccccuuccuggacgucuacuaccacgagaacaacaagagcaggauggaaagcgagcuccggguguacagcagcgccaacaacugcaccuucgaguacgugucccagccuuuccugauggaccuggaaggcaagcagggcaacuucaagaaccugcgcgaguucguguuuaagaacaucgacggcuacuucaagaucuacagcaagcacaccccugugaaccucggccgggaucugccucagggcuucucugcucuggaaccccugguggaucugcccaucggcaucaacaucacccgguuucagacacugcuggcccugcacagaagcuaccugacaccuggcgauagcagcagcagcuggacagcuggugccgccgcuuacuaugugggcuaccugcagccuagaaccuuccugcugaaguacaacgagaacggcaccaucaccgacgccguggauugugcucuggauccucugagcgagacaaagugcacccugaaguccuucaccguggaaaagggcaucuaccagaccagcaacuuccgggugcagcccaccgaauccaucgugcgguuccccaauaucaccaaucugugccccuuccacgagguguucaaugccaccagauucgccucuguguacgccuggaaccggaagcggaucagcaauugcguggccgacuacuccgugcuguacaacuucgcccccuucuucgcauucaagugcuacggcguguccccuaccaagcugaacgaccugugcuucacaaacguguacgccgacagcuucgugauccggggaaacgaagugucacagauugccccuggacagacaggcaacaucgccgacuacaacuacaagcugcccgacgacuucaccggcugugugauugccuggaacagcaacaagcuggacuccaaagucagcggcaacuacaauuaccuguaccggcuguuccggaaguccaagcugaagcccuucgagcgggacaucuccaccgagaucuaucaggccggcaacaagccuuguaacggcguggcaggcuucaacugcuacuucccacugcaguccuacggcuuuaggcccacauacggcgugggccaccagcccuacagagugguggugcugagcuucgaacugcugcaugccccugccacagugugcggcccuaagaaaagcaccaaucucgugaagaacaaaugcgugaacuucaacuucaacggccugaccggcaccggcgugcugacagagagcaacaagaaguuccugccauuccagcaguuuggccgggauaucgccgauaccacagacgccguuagagauccccagacacuggaaauccuggacaucaccccuugcagcuucggcggagugucugugaucaccccuggcaccaacaccagcaaucagguggcagugcuguaccagggcgugaacuguaccgaagugcccguggccauucacgccgaucagcugacaccuacauggcggguguacuccaccggcagcaauguguuucagaccagagccggcugucugaucggagccgaguacgugaacaauagcuacgagugcgacauccccaucggcgcuggaaucugcgccagcuaccagacacagacaaagagccaccggagagccagaagcguggccagccagagcaucauugccuacacaaugucucugggcgccgagaacagcguggccuacuccaacaacucuaucgcuagccccaccaacuucaccaucagcgugaccacagagauccugccuguguccaugaccaagaccagcguggacugcaccauguacaucugcggcgauuccaccgagugcuccaaccugcugcugcaguacggcagcuucugcacccagcugaaaagagcccugacagggaucgccguggaacaggacaagaacacccaagagguguucgcccaagugaagcagaucuacaagaccccuccuaucaaguacuucggcggcuucaauuucagccagauucugcccgauccuagcsagcccagcaagcggagcuucaucgaggaccugcuguucaacaaagugacacuggccgacgccggcuucaucaagcaguauggcgauugucugggcgacauugccgccagggaucugauuugcgcccagaaguuuaacggacugacagugcugccuccucugcugaccgaugagaugaucgcccaguacacaucugcccugcuggccggcacaaucacaagcggcuggacauuuggagcaggcgccgcucugcagauccccuuugcuaugcagauggccuaccgguucaacggcaucggagugacccagaaugugcuguacgagaaccagaagcugaucgccaaccaguucaacagcgccaucggcaagauccaggacagccugagcagcacagcaagcgcccugggaaagcugcaggacguggucaaccacaaugcccaggcacugaacacccuggucaagcagcuguccuccaaguucggcgccaucagcucugugcugaacgauauccugagcagacuggacccuccugaggccgaggugcagaucgacagacugaucacaggcagacugcagagccuccagacauacgugacccagcagcugaucagagccgccgagauuagagccucugccaaucuggccgccaccaagaugucugagugugugcugggccagagcaagagaguggacuuuugcggcaagggcuaccaccugaugagcuucccucagucugccccucacggcgugguguuucugcacgugacauaugugcccgcucaagagaagaauuucaccaccgcuccagccaucugccacgacggcaaagcccacuuuccuagagaaggcguguucguguccaacggcacccauugguucgugacacagcggaacuucuacgagccccagaucaucaccaccgacaacaccuucgugucuggcaacugcgacgucgugaucggcauugugaacaauaccguguacgacccucugcagcccgagcuggacagcuucaaagaggaacuggacaaquacuuuaagaaccacacaagccccgacguggaccugggcgauaucagcggaaucaaugccagcgucgugaacauccagaaagagaucgaccggcugaacgagguggccaagaaucugaacgagagccugaucgaccugcaagaacuggggaaguacgagcaguacaucaaguggcccugguacaucuggcugggcuuuaucgccggacugauugccaucgugauggucacaaucaugcuguguugcaugaccagcugcuguagcugccugaagggcuguuguagcuguggcagcugcugcaaguucgacgaggacgauucugagcccgugcugaagggcgugaaacugcacuacacaugaugacucgagcugguacugcaugcacgcaaugcuagcugccccuuucccguccuggguaccccgagucucccccgaccucgggucccagguaugcucccaccuccaccugccccacucaccaccucugcuaguuccagacaccucccaagcacgcagcaaugcagcucaaaacgcuuagccuagccacacccccacgggaaacagcagugauuaaccuuuagcaauaaacgaaaguuuaacuaagcuauacuaaccccaggguuggucaauuucgugccagccacacccuggagcuagcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagcauaugacuaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 84Full length DNA constructagaataaactagtattcttctggtccccacagactcagagagaacccgccaccatgttcgtgttccencoding a SARS-CoV-2 Stggtgctgctgcctctggtgtccagccagtgtgtgaacctgatcaccagaacacagtcatacaccaprotein from an OmicronacagctttaccagaggcgtgtactaccccgacaaggtgttcagatccagcgtgctgcactctacccBA.2.75 variantaggacctgttcctgcctttcttcagcaacgtgacctggttccacgccatccacgtgtccggcaccaatggcaccaagagattcgacaaccccgtgctgcccttcaacgacggggtgtactttgccagcaccgagaagtccaacatcatcagaggctggatcttcggcaccacactggacagcaagacccagagcctgctgatcgtgaacaacgccaccaacgtggtcatcaaagtgtgcgagttccagttctgcaacgaccccttcctggacgtctactaccacgagaacaacaagagcaggatggaaagcgagctccgggtgtacagca gcgccaacaactgcaccttcgagtacgtgtcccagcctttcctgatggacctggaaggcaagcagggcaacttcaagaacctgcgcgagttcgtgtttaagaacatcgacggctacttcaagatctacagcaagcacacccctgtgaacctcggccgggatctgcctcagggcttctctgctctggaacccctggtggatctgcccatcggcatcaacatcacccggtttcagacactgctggccctgcacagaagctacctgacacctggcgatagcagcagcagctggacagctggtgccgccgcttactatgtgggctacctgcagcctagaaccttcctgctgaagtacaacgagaacggcaccatcaccgacgccgtggattgtgctctggatcctctgagcgagacaaagtgcaccctgaagtccttcaccgtggaaaagggcatctaccagaccagcaacttccgggtgcagcccaccgaatccatcgtgcggttccccaatatcaccaatctgtgccccttccacgaggtgttcaatgccaccagattcgcctctgtgtacgcctggaaccggaagcggatcagcaattgcgtggccgactactccgtgctgtacaacttcgcccccttcttcgcattcaagtgctacggcgtgtcccctaccaagctgaacgacctgtgcttcacaaacgtgtacgccgacagcttcgtgatccggggaaacgaagtgtcacagattgcccctggacagacaggcaacatcgccgactacaactacaagctgcccgacgacttcaccggctgtgtgattgcctggaacagcaacaagctggactccaaagtcagcggcaactacaattacctgtaccggctgttccggaagtccaagctgaagcccttcgagcgggacatctccaccgagatctatcaggccggcaacaagccttgtaacggcgtggcaggcttcaactgctacttcccactgcagtcctacggctttaggcccacatacggcgtgggccaccagccctacagagtggtggtgctgagcttcgaactgctgcatgcccctgccacagtgtgcggccctaagaaaagcaccaatctcgtgaagaacaaatgcgtgaacttcaacttcaacggcctgaccggcaccggcgtgctgacagagagcaacaagaagttcctgccattccagcagtttggccgggatatcgccgataccacagacgccgttagagatccccagacactggaaatcctggacatcaccccttgcagcttcggcggagtgtctgtgatcacccctggcaccaacaccagcaatcaggtggcagtgctgtaccagggcgtgaactgtaccgaagtgcccgtggccattcacgccgatcagctgacacctacatggcgggtgtactccaccggcagcaatgtgtttcagaccagagccggctgtctgatcggagccgagtacgtgaacaatagctacgagtgcgacatccccatcggcgctggaatctgcgccagctaccagacacagacaaagagccaccggagagccagaagcgtggccagccagagcatcattgcctacacaatgtctctgggcgccgagaacagcgtggcctactccaacaactctatcgctatccccaccaacttcaccatcagcgtgaccacagagatcctgcctgtgtccatgaccaagaccagcgtggactgcaccatgtacatctgcggcgattccaccgagtgctccaacctgctgctgcagtacggcagcttctgcacccagctgaaaagagccctgacagggatcgccgtggaacaggacaagaacacccaagaggtgttcgcccaagtgaagcagatctacaagacccctcctatcaagtacttcggcggcttcaatttcagccagattctgcccgatcctagcaagcccagcaagcggagcttcatcgaggacctqctgttcaacaaagtgacactggccgacgccggcttcatcaagcagtatggcgattgtctgggcgacattgccgccagggatctgatttgcgcccagaagtttaacggactgacagtgctgcctcctctgctgaccgatgagatgatcgcccagtacacatctgccctgctggccggcacaatcacaagcggctggacatttggagcaggcgccgctctgcagatcccctttgctatgcagatggcctaccggttcaacggcatcggagtgacccagaatgtgctgtacgagaaccagaagctgatcgccaaccagttcaacagcgccatcggcaagatccaggacagcctgagcagcacagcaagcgccctgggaaagctgcaggacgtggtcaaccacaatgcccaggcactgaacaccctggtcaagcagctgtcctccaagttcggcgccatcagctctgtgctgaacgatatcctgagcagactggaccctcctgaggccgaggtgcagatcgacagactgatcacaggcagactgcagagcctccagacatacgtgacccagcagctgatcagagccgccgagattagagcctctgccaatctggccgccaccaagatgtctgagtgtgtgctgggccagagcaagagagtggacttttgcggcaagggctaccacctgatgagcttccctcagtctgcccctcacggcgtggtgtttctgcacgtgacatatgtgcccgctcaagagaagaatttcaccaccgctccagccatctgccacgacggcaaagcccactttcctagagaaggcgtgttcgtgtccaacggcacccattggttcgtgacacagcggaacttctacgagccccagatcatcaccaccgacaacaccttcgtgtctggcaactgcgacgtcgtgatcggcattgtgaacaataccgtgtacgaccctctgcagcccgagctggacagcttcaaagaggaactggacaagtactttaagaaccacacaagccccgacgtggacctgggcgatatcagcggaatcaatgccagcgtcgtgaacatccagaaagagatcgaccggctgaacgaggtggccaagaatctgaacgagagcctgatcgacctgcaagaactggggaagtacgagcagtacatcaagtggccctggtacatctggctgggctttatcgccggactgattaccatcgtgatggtcacaatcatgctgtgttgcatgaccagctgctgtagctgcctgaagggctgttgtagctgtggcagctgctgcaagttcgacgaggacgattctgagcccgtgctgaagggcgtgaaactgcactacacatgatgactcgagctggtactgcatgcacgcaatgctagctgcccctttcccgtcctgggtaccccgagtctcccccgacctcgggtcccaggtatgctcccacctccacctgccccactcaccacctctgctagttccagacacctcccaagcacgcagcaatgcagctcaaaacgcttagcctagccacacccccacgggaaacagcagtgattaacctttagcaataaacgaaagtttaactaagctatactaaccccagggttggtcaatttcgtgccagccacaccctggagctagcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagcatatgactaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa

TABLE 13Sequence of one embodiment of an exemplary Omicron BA.2.75.2-specific RNA vaccineSEQ ID NO. Brief Description Sequence 85 Amino acid sequence of RNA-MFVFLVLLPLVSSQCVNLITRTQSYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHencoded SARS-CoV-2 S proteinVSGTNGTKRFDNPVLPENDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFfrom an Omicron BA.2.75.2CNDPFLDVYYHENNKSRMESELRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFvariant (with PRO mutations atKIYSKHTPVNLGRDLPQGFSALEPLVDLPIGINITREQTLLALHRSYLTPGDSSSSWTAGAAAYYVpositions corresponding toGYLQPRTELLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITK986P and V987P of SEQ ID NO:NLCPFHEVFNATTFASVYAWNRKRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADS1; i.e., PRO mutations atFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVSGNYNYLYRLFRKSKLKPFEpositions 983 and 984 of SEQRDISTELYQAGNKPCNGVAGSNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTID NO: 85)NLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEYVNNSYECDIPIGAGICASYQTQTKSHRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLKRALTGIAVEQDKNTQEVFAQVKQTYKTPPIKYFGGFNFSQILPDPSKPSKRSFIEDLLENKVTLADAGFIKQYGDCLGDIAARDLICAQKENGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRENGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNHNAQALNTLVKQLSSKFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVEVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLINLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT 86 RNA sequence encoding a SARS-auguucguguuccuggugcugcugccugugguguccagccagugugugaaccugaucaccagaacaCoV-2 S protein from ancagucauacaccaacagcuuuaccagaggcguguacuaccccgacaagguguucagauccagcgugOmicron BA.2.75.2 variantcugcacucuacccaggaccuguuccugccuuucuucagcaacgugaccugguuccacgccauccacguguccggcaccaauggcaccaagagauucgacaaccccgugcugcccuucaacgacgggguguacuuugccagcaccgagaaguccaacaucaucagaggcuggaucuucggcaccacacuggacagcaagacccagagccugcugaucgugaacaacgccaccaacguggucaucaaagugugcgaguuccaguucugcaacgaccccuuccuggacqucuacuaccacgagaacaacaagagcaggauggaaagcgagcuccggguguacagcagcgccaacaacugcaccuucgaguacgugucccagccuuuccugauggaccuggaaggcaagcagggcaacuucaagaaccugcgcgaguucguguuuaagaacaucgacggcuacuucaagaucuacagcaagcacaccccugugaaccucggccgggaucugccucagggcuucucugcucuggaaccccugguggaucugcccaucggcaucaacaucacccgguuucagacacugcuggcccugcacagaagcuaccugacaccuggcgauagcagcagcagcuggacagcuggugccgccgcuuacuaugugggcuaccugcagccuagaaccuuccugcugaaguacaacgagaacggcaccaucaccgacgccguggauugugcucuggauccucugagcgagacaaagugcacccugaaguccuucaccguggaaaagggcaucuaccagaccagcaacuuccgggugcagcccaccgaauccaucgugcgguuccccaauaucaccaaucugugccccuuccacgagguguucaaugccaccaccuucgccucuguguacgccuggaaccggaagcggaucagcaauugcguggccgacuacuccgugcuguacaacuucgcccccuucuucgcauucaagugcuacggcguguccccuaccaagcugaacgaccugugcuucacaaacguguacgccgacagcuucgugauccggggaaacgaagugucacagauugccccuggacagacaggcaacaucgccgacuacaacuacaagcugcccgacgacuucaccggcugugugauugccuggaacagcaacaagcuggacuccaaagucagcggcaacuacaauuaccuguaccggcuguuccggaaguccaagcugaagcccuucgagcgggacaucuccaccgagaucuaucaggccggcaacaagccuuguaacggcguggcaggcagcaacugcuacuucccacugcaguccuacggcuuuaggcccacauacggcgugggccaccagcccuacagagugguggugcugagcuucgaacugcugcaugccccugccacagugugcggcccuaagaaaagcaccaaucucgugaagaacaaaugcgugaacuucaacuucaacggccugaccggcaccggcgugcugacagagagcaacaagaaguuccugccauuccagcaguuuggccgggauaucgccgauaccacagacgccguuagagauccccagacacuggaaauccuggacaucaccccuugcagcuucggcggagugucugugaucaccccuggcaccaacaccagcaaucagguggcagugcuguaccagggcgugaacuguaccgaagugcccguggccauucacgccgaucagcugacaccuacauggcgguguacucccaccggcagcaauguguuucagaccagagccggcugucugaucggagccgaguacgugaacaauagcuacgagugcgacauccccaucggcgcuggaaucugcgccagcuaccagacacagacaaagagccaccggagagccagaagcguggccagccagagcaucauugccuacacaaugucucugggcgccgagaacgacguggccuacuccaacaacucuaucgcuauccccaccaacuucaccaucagcgugaccacagagauccugccuguguccaugaccaagaccagcguggacugcaccauguacaucugcggcgauuccaccgagugcuccaaccugcugcugcaguacggcagcuucugcacccagcugaaaagagcccugacagggaucgccguggaacaggacaagaacacccaagagguguucgcccaagugaagcagaucuacaagaccccuccuaucaaguacuucggcggcuucaauuucagccagauucugcccgauccuagcaagcccagcaagcggagcuucaucgaggaccugcuguucaacaaagugacacuggccgacgccggcuucaucaagcaguauggcgauugucugggcgacauugccgccagggaucugauuugcgcccagaaguuuaacggacugacagugcugccuccucugcugaccgaugagaugaucgcccaguacacaucugcccugcuggccggcacaaucacaagcggcuggacauuuggagcaggcgccgcucugcagauccccuuugcuaugcagauggccuaccgguucaacggcaucggagugacccagaaugugcuguacgagaaccagaagcugaucgccaaccaguucaacagcgccaucggcaagauccaggacagccugagcagcacagcaagcgcccugggaaagcugcaggacguggucaaccacaaugcccaggcacugaacacccuggucaagcagcuguccuccaaguucggcgccaucagcucugugcugaacgauauccugagcagacuggacccuccugaggccgaggugcagaucgacagacugaucacaggcagacugcagagccuccagacauacgugacccagcagcugaucagagccgccgagauuagagccucugccaaucuggccgccaccaagaugucugagugugugcugggccagagcaagagaguggacuuuugcggcaagggcuaccaccugaugagcuucccucagucugccccucacggcgugguguuucugcacgugacauaugugcccgcucaagagaagaauuucaccaccgcuccagccaucugccacgacggcaaagcccacuuuccuagagaaggcguguucguguccaacggcacccauugguucgugacacagcggaacuucuacgagccccagaucaucaccaccgacaacaccuucgugucuggcaacugcgacgucgugaucggcauugugaacaauaccguguacgacccucugcagcccgagcuggacagcuucaaagaggaacuggacaaguacuuuaagaaccacacaagccccgacguggaccugggcgauaucagcggaaucaaugccagcgucgugaacauccagaaagagaucgaccggcugaacgagguggccaagaaucugaacgagagccugaucaaccugcaagaacuggggaaquacgagcaguacaucaaguggcccugguacaucuggcugggcuuuaucgccggacugauugccaucgugauggucacaaucaugcuguguugcaugaccagcugcuguagcugccugaagggcuguuguagcuguggcagcugcugcaaguucgacgaggacgauucugagcccgugcugaagggcgugaaacugcacuacacaugauga 87DNA sequence encoding a SARS-atgttcgtgttcctggtgctgctgcctctggtgtccagccagtgtgtgaacctgatcaccagaacaCoV-2 S protein from ancagtcatacaccaacagctttaccagaggcgtgtactaccccgacaaggtgttcagatccagcgtgOmicron BA.2.75.2 variantctgcactctacccaggacctgttcctgcctttcttcagcaacgtgacctggttccacgccatccacgtgtccggcaccaatggcaccaagagattcgacaaccccgtgctgcccttcaacgacggggtgtactttgccagcaccgagaagtccaacatcatcagaggctggatcttcggcaccacactggacagcaagacccagagcctgctgatcgtgaacaacgccaccaacgtggtcatcaaagtgtgcgagttccagttctgcaacgaccccttcctggacgtctactaccacgagaacaacaagagcaggatggaaagcgagctccgggtgtacagcagcgccaacaactgcaccttcgagtacgtgtcccagcctttcctgatggacctggaaggcaagcagggcaacttcaagaacctgcgcgagttcgtgtttaagaacatcgacggctacttcaagatctacagcaagcacacccctgtgaacctcggccgggatctgcctcagggcttctctgctctggaacccctggtggatctgcccatcggcatcaacatcacccggtttcagacactgctggccctgcacagaagctacctgacacctggcgatagcagcagcagctggacagctggtgccgccgcctactatgtgggctacctgcagcctagaaccttcctgctgaagtacaacgagaacggcaccatcaccgacgccgtggattgtgctctggatcctctgagcgagacaaagtgcaccctgaagtccttcaccgtggaaaagggcatctaccagaccagcaacttccgggtgcagcccaccgaatccatcgtgcggttccccaatatcaccaatctgtgccccttccacgaggtgttcaatgccaccaccttcgcctctgtgtacgcctggaaccggaagcggatcagcaattgcgtggccgactactccgtgctgtacaacttcgcccccttcttcgcattcaagtgctacggcgtgtcccctaccaagctgaacgacctgtgcttcacasacgtgtacgccgacagcttcgtgatccggggaaacgaagtgtcacagattgcccctggacagacaggcaacatcgccgactacaactacaagctgcccgacgacttcaccggctgtgtgattgcctggaacagcaacaagctggactccaaagtcagcggcaactacaattacctgtaccggctgttccggaagtccaagctgaagcccttcgagcgggacatctccaccgagatctatcaggccggcaacaagccttgtaacggcgtggcaggcagcaactgctacttcccactgcagtcctacggctttaggcccacatacggcgtgggccaccagccctacagagtggtggtgctgagcttcgaactgctgcatgcccctgccacagtgtgcggccctaagaaaagcaccaatctcgtgaagaacaaatgcgtgaacttcaacttcaacggcctgaccggcaccggcgtgctgacagagagcaacaagaagttcctgccattccagcagtttggccgggatatcgccgataccacagacgccgttagagatccccagacactggaaatcctggacatcaccccttgcagcttcggcggagtgtctgtgatcacccctggcaccaacaccagcaatcaggtggcagtgctgtaccagggcgtgaactgtaccgaagtgcccgtggccattcacgccgatcagctgacacctacatggcgggtgtactccaccggcagcaatgtgtttcagaccagagccggctgtctgatcggagccgagtacgtgaacaatagctacgagtgcgacatccccatcggcgctggaatctgcgccagctaccagacacagacaaagagccaccggagagccagaagcgtggccagccagagcatcattgcctacacaatgtctctgggcgccgagaacagcgtggcctactccaacaactctatcgctatccccaccaacttcaccatcagcgtgaccacagagatcctgcctgtgtccatgaccaagaccagcgtggactgcaccatgtacatctgcggcgattccaccgagtgctccaacctgctgctgcagtacggcagcttctgcacccagctgaaaagagccctgacagggatcgccgtggaacaggacaagaacacccaagaggtcttcgcccaagtgaagcagatctacaagacccctcctatcaagtacttcggcggcttcaatttcagccagattctgcccgatcctagcaagcccagcaagcggagcttcatcgaggacctgctgttcaacaaagtgacactggccgacgccggcttcatcaagcagtatggcgattgtctgggcgacattgccgccagggatctgatttgcgcccagaagtttaacggactgacagtgctgcctcctctgctgaccgatgagatgatcgcccagtacacatctgccctgctggccggcacaatcacaagcggctggacatttggagcaggcgccgctctgcagatcccctttgctatgcagatggcctaccggttcaacggcatcggagtgacccagaatgtgctgtacgagaaccagaagctgatcgccaaccagttcaacagcgccatcggcaagatccaggacagcctgagcagcacagcaagcgccctgggaaagctgcaggacgtggtcaaccacaatgcccaggcactgaacaccctggtcaagcagctgtcctccaagttcggcgccatcagctctgtgctgaacgatatcctgagcagactggaccctcctgaggccgaggtgcagatcgacagactgatcacaggcagactgcagagcctccagacatacgtgacccagcagctgatcagagccgccgagattagagcctctgccaatctggccgccaccaagatgtctgagtgtgtgctgggccagagcaagagagtggacttttgcggcaagggctaccacctgatgagcttccctcagtctgcccctcacggcgtggtgtttctgcacgtgacatatgtgcccgctcaagagaagaatttcaccaccgctccagccatctgccacgacggcaaagcccactttcctagagaaggcgtgttcgtgtccaacggcacccattggttcgtgacacagcggaacttctacgagccccagatcatcaccaccgacaacaccttcgtgtctggcaactgcgacgtcgtgatcggcattgtgaacaataccgtgtacgaccctctgcagcccgagctggacagcttcaaagaggaactggacaagtactttaagaaccacacaagccccgacgtggacctgggcgatatcagcggaatcaatgccagcgtcgtgaacatccagaaagagatcgaccggctgaacgaggtggccaagaatctgaacgagagcctgatcaacctgcaagaactggggaagtacgagcagtacatcaagtggccctggtacatctggctgggctttatcgccggactgattgccatcgtgatggtcacaatcatgctgtgttgcatgaccagctgctgtagctgcctgaagggctgttgtagctgtggcagctgctgcaagttcgacgaggacgattctgagcccgtgctgaagggcgtgaaactgcactacacatgatga 88Full length RNA constructagaauaaacuaguauucuucugguccccacagacucagagagaacccgccaccauguucguguuccencoding a SARS-CoV-2 Suggugcugcugccucugguguccagccagugugugaaccugaucaccagaacacagucauacaccaprotein from an OmicronacagcuuuaccagaggcguguacuaccccgacaagguguucagauccagcgugcugcacucuacccBA.2.75.2 variantaggaccuquuccugccuuucuucagcaacgugaccugguuccacgccauccacguguccggcaccaauggcaccaagagauucgacaaccccgugcugcccuucaacgacgggguguacuuugccagcaccgagaaguccaacaucaucagaggcuggaucuucggcaccacacuggacagcaagacccagagccugcugaucgugaacaacgccaccaacguggucaucaaagugugcgaguuccaguucugcaacgaccccuuccuggacgucuacuaccacgagaacaacaagagcaggauggaaagcgagcuccggguguacagcagcgccascaacugcaccuucgaquacgugucccagccuuuccugauggaccuggaaggcaagcagggcaacuucaagaaccugcgcgaguucguguuusagaacaucgacggcuacuucaagaucuacagcaagcacaccccugugaaccucggccgggaucugccucagggcuucucugcucuggaaccccugguggaucugcccaucggcaucaacaucacccgguuucagacacugcuggcccugcacagaagcuaccugacaccuggcgauagcagcagcagcuggacagcuggugccgccgcuuacuaugugggcuaccugcagccuagaaccuuccugcugaaguacaacgagaacggcaccaucaccgacgccguggauugugcucuggauccucugagcgagacaaagugcacccugaaguccuucaccguggaaaagggcaucuaccagaccagcaacuuccgggugcagcccaccgaauccaucgugcgguuccccaauaucaccaaucugugccccuuccacgagguguucaaugccaccaccuucgccucuguguacgccuggaaccggaagcggaucagcaauugcguggccgacuacuccgugcuguacaacuucgcccccuucuucgcauucaagugcuacggcguguccccuaccaagcugaacgaccugugcuucacaaacguguacgccgacagcuucgugauccggggaaacgaagugucacagauugccccuggacagacaggcaacaucgccgacuacaacuacaagcugcccgacgacuucaccggcugugugauugccuggaacagcaacaagcuggacuccaaagucagcggcaacuacaauuaccuguaccggcuguuccggaaguccaagcugaagcccuucgagcgggacaucuccaccgagaucuaucaggccggcaacaagccuuguaacggcguggcaggcagcaacugcuacuucccacugcaguccuacggcuuuaggcccacauacggcgugggccaccagcccuacagagugguggugcugagcuucgaacugcugcaugccccugccacagugugcggcccuaagaaaagcaccaaucucgugaagaacaaaugcgugaacuucaacuucaacggccugaccggcaccggcgugcugacagagagcaacaagaaguuccugccauuccagcaguuuggccgggauaucgccgauaccacagacgccguuagagauccccagacacuggaaauccuggacaucaccccuugcagcuucggcggagugucugugaucaccccuggcaccaacaccagcaaucagguggcagugcuguaccagggcgugaacuguaccgaagugcccguggccauucacgccgaucagcugacaccuacauggcggguguacuccaccggcagcaauguguuucagaccagagccggcugucugaucggagccgaguacgugaacaauagcuacgagugcgacauccccaucggcgcuggaaucugcgccagcuaccagacacagacaaagagccaccggagagccagaagcguggccagccagagcaucauugccuacacaaugucucugggcgccgagaacagcguggccuacuccaacaacucuaucgcuauccccaccaacuucaccaucagcgugaccacagagauccugccuguguccaugaccaagaccagcguggacugcaccauguacaucugcggcgauuccaccgagugcuccaaccugcugcugcaguacggcagcuucugcacccagcugaaaagagcccugacagggaucgccguggaacaggacaagaacacccaagagguguucgcccaagugaagcagaucuacaagaccccuccuaucaaguacuucggcggcuucaauuucagccagauucugcccgauccuagcaagcccagcaagcggagcaucaucgaggaccugcuguucaacaaagugacacuggccgacgccggcuucaucaagcaguauggcgauugucugggcgacauugccgccagggaucugauuugcgcccagaaguuuaacggacugacagugcugccuccucugcugaccgaugagaugaucgcccaguacacaucugcccugcuggccggcacaaucacaagcggcuggacauuuggagcaggcgccgcucugcagauccccuuugcuaugcagauggccuaccgguucaacggcaucggagugacccagaaugugcuguacgagaaccagaagcugaucgccaaccaguucaacagcgccaucggcaagauccaggacagccugagcagcacagcaagcgcccugggaaagcugcaggacguggucaaccacaaugcccaggcacugaacacccuggucaagcagcuguccuccaaguucggcgccaucagcucugugcugaacgauauccugagcagacuggacccuccugaggccgaggugcagaucgacagacugaucacaggcagacugcagagccuccagacauacgugacccagcagcugaucagagccgccgagauuagagccucugccaaucuggccgccaccaagaugucugagugugugcugggccagagcaagagaguggacuuuugcggcaagggcuaccaccugaugagcuucccucagucugccccucacggcgugguguuucugcacgugacauaugugcccgcucaagagaagaauuucaccaccgcuccagccaucugccacgacggcaaagcccacuuuccuagagaaggcguguucguguccaacggcacccauugguucgugacacagcggaacuucuacgagccccagaucaucaccaccgacaacaccuucgugucuggcaacugcgacgucgugaucggcauugugaacaauaccguguacgacccucugcagcccgagcuggacagcuucaaagaggaacuggacaaquacuuuaagaaccacacaagccccgacguggaccugggcgauaucagcggaaucaaugccagcgucgugaacauccagaaagagaucgaccggcugaacgagguggccaagaaucugaacgagagccugaucaaccugcaagaacuggggaaguacgagcaguacaucaaguggcccugquacaucuggcugggcuuuaucgccggacugauugccaucgugauggucacaaucaugcuguguugcaugaccagcugcuguagcugccugaagggcuguuguagcuguggcagcugcugcaaguucgacgaggacgauucugagcccgugcugaagggcgugaaacugcacuacacaugaugacucgagcugguacugcaugcacgcaaugcuagcugccccuuucccguccuggguaccccgagucucccccgaccucgggucccagguaugcucccaccuccaccugccccacucaccaccucugcuaguuccagacaccucccaagcacgcagcaaugcagcucaaaacgcuuagccuagccacacccccacgggaaacagcagugauuaaccuuuagcaauaaacgaaaguuuaacuaagcuauacuaaccccaggguuggucaauuucgugccagccacacccuggagcuagcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagcauaugacuaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 89Full length DNA constructagaataaactagtattcttctggtccccacagactcagagagaacccgccaccatgttcgtgttccencoding a SARS-CoV-2 Stggtgctgctgcctctggtgtccagccagtgtgtgaacctgatcaccagaacacagtcatacaccaprotein from an OmicronacagctttaccagaggcgtgtactaccccgacaaggtgttcagatccagcgtgctgcactctacccBA.2.75.2 variantaggacctgttcctgcctttcttcagcaacgtgacctggttccacgccatccacgtgtccggcaccaatggcaccaagagattcgacaaccccgtgctgcccttcaacgacggggtgtactttgccagcaccgagaagtccaacatcatcagaggctggatcttcggcaccacactggacagcaagacccagagcctgctgatcgtgaacaacgccaccaacgtggtcatcaaagtgtgcgagttccagttctgcaacgaccccttcctggacgtctactaccacgagaacaacaagagcaggatggaaagcgagctccgggtgtacagcagcgccaacaactgcaccttcgagtacgtgtcccagcctttcctgatggacctggaaggcaagcagggcaacttcaagaacctgcgcgagttcgtgtttaagaacatcgacggctacttcaagatctacagcaagcacacccctgtgaacctcggccgggatctgcctcagggcttctctgctctggaacccctggtggatctgcccatcggcatcaacatcacccggtttcagacactgctggccctgcacagaagctacctgacacctggcgatagcagcagcagctggacagctggtgccgccgcttactatgtgggctacctgcagcctagaaccttcctgctgaagtacaacgagaacggcaccatcaccgacgccgtggattgtgctctggatcctctgagcgagacaaagtgcaccctgaagtccttcaccgtggaaaagggcatctaccagaccagcaacttccgggtgcagcccaccgaatccatcgtgcggttccccaatatcaccaatctgtgccccttccacgaggtgttcaatgccaccaccttcgcctctgtgtacgcctggaaccggaagcggatcagcaattgcgtggccgactactccgtgctgtacaacttcgcccccttcttcgcattcaagtgctacggcgtgtcccctaccaagctgaacgacctgtgcttcacaaacgtgtacgccgacagcttcgtgatccggggaaacgaagtgtcacagattgcccctggacagacaggcaacatcgccgactacaactacaagctgcccgacgacttcaccggctgtgtgattgcctggaacagcaacaagctggactccaaagtcagcggcaactacaattacctgtaccggctgttccggaagtccaagctgaagcccttcgagcgggacatctccaccgagatctatcaggccggcaacaagccttgtaacggcgtggcaggcagcaactgctacttcccactgcagtcctacggctttaggcccacatacggcgtgggccaccagccctacagagtggtggtgctgagcttcgaactgctgcatgcccctgccacagtgtgcggccctaagaaaagcaccaatctcgtgaagaacaaatgcgtgaacttcaacttcaacggcctgaccggcaccggcgtgctgacagagagcaacaagaagttcctgccattccagcagtttggccgggatatcgccgataccacagacgccgttagagatccccagacactggaaatcctggacatcaccccttgcagcttcggcggagtgtctgtgatcacccctggcaccaacaccagcaatcaggtggcagtgctgtaccagggcgtgaactgtaccgaagtgcccgtggccattcacgccgatcagctgacacctacatggcgggtgtactccaccggcagcaatgtgtttcagaccagagccggctgtctgatcggagccgagtacgtgaacaatagctacgagtgcgacatccccatcggcgctggaatctgcgccagctaccagacacagacaaagagccaccggagagccagaagcgtggccagccagagcatcattgcctacacaatgtctctgggcgccgagaacagcgtggcctactccaacaactctatcgctatccccaccaacttcaccatcagcgtgaccacagagatcctgcctgtgtccatgaccaagaccagcgtggactgcaccatgtacatctgcggcgattccaccgagtgctccaacctgctgctgcagtacggcagcttctgcacccagctgaaaagagccctgacagggatcgccgtggaacaggacaagaacacccaagaggtgttcgcccaagtgaagcagatctacaagacccctcctatcaagtacttcggcggcttcaatttcagccagattctgcccgatcctagcaagcccagcaagcggagcttcatcgaggacctgctgttcaacaaagtgacactggccgacgccggcttcatcaagcagtatggcgattgtctgggcgacattgccgccagggatctgatttgcgcccagaagtttaacggactgacagtgctgcctcctctgctgaccgatgagatgatcgcccagtacacatctgccctgctggccggcacaatcacaagcggctggacatttggagcaggcgccgctctgcagatcccctttgctatgcagatggcctaccggttcaacggcatcggagtgacccagaatgtgctgtacgagaaccagaagctgatcgccaaccagttcaacagcgccatcggcaagatccaggacagcctgagcagcacagcaagcgccctgggaaagctgcaggacgtggtcaaccacaatgcccaggcactgaacaccctggtcaagcagctgtcctccaagttcggcgccatcagctctgtgctgaacgatatcctgagcagactggaccctcctgaggccgaggtgcagatcgacagactgatcacaggcagactgcagagcctccagacatacgtgacccagcagctgatcagagccgccgagattagagcctctgccaatctggccgccaccaagatgtctgagtgtgtgctgggccagagcaagagagtggacttttgcggcaagggctaccacctgatgagcttccctcagtctgcccctcacggcgtggtgtttctgcacgtgacatatgtgcccgctcaagagaagaatttcaccaccgctccagccatctgccacgacggcaaagcccactttcctagagaaggcgtgttcgtgtccaacggcacccattggttcgtgacacagcggaacttctacgagccccagatcatcaccaccgacaacaccttcgtgtctggcaactgcgacgtcgtgatcggcattgtgaacaataccgtgtacgaccctctgcagcccgagctggacagcttcaaagaggaactggacaagtactttaagaaccacacaagccccgacgtggacctgggcgatatcagcggaatcaatgccagcgtcgtgaacatccagaaagagatcgaccggctgaacgaggtggccaagaatctgaacgagagcctgatcaacctgcaagaactggggaagtacgagcagtacatcaagtggccctggtacatctggctgggctttatcgccggactgattgccatcgtgatggtcacaatcatgctgtgttgcatgaccagctgctgtagctgcctgaagggctgttgtagctgtggcagctgctgcaagttcgacgaggacgattctgagcccgtgctgaagggcgtgaaactgcactacacatgatgactcgagctggtactgcatgcacgcaatgctagctgcccctttcccgtcctgggtaccccgagtctcccccgacctcgggtcccaggtatgctcccacctccacctgccccactcaccacctctgctagttccagacacctcccaagcacgcagcaatgcagctcaaaacgcttagcctagccacacccccacgggaaacagcagtgattaacctttagcaataaacgaaagtttaactaagctatactaaccccagggttggtcaatttcgtgccagccacaccctggagctagcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagcatatgactaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa

TABLE 14Sequence of one embodiment of an exemplary Omicron BA.4.6/BF.7-specific RNA vaccineSEQ   ID NO. Brief Description Sequence 90 Amino acid sequence of RNA-MFVFLVLLPLVSSQCVNLITRTQSYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAISencoded SARS-Cov-2 S proteinGTNGTKRFDNPVLPENDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNfrom an Omicron BA.4.6/BF.7DPFLDVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIvariant (with PRO mutations YSKHTPINLGRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYat positions corresponding CPFDEVFNATTFASVYAWNRKRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVto K986P and V987P of SEQ  IRGNEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVGGNYNYRYRLFRKSNLKPFERDID NO: 1; i.e., PRO muta-ISTEIYQAGNKPCNGVAGVNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLtions at positions 981 and   VKNKCVNFNFNGLTGTGVLTESNKKELPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVIT982 of SEQ ID NO: 90)PGTNTSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEYVNNSYECDIPIGAGICASYQTQTKSHRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLKRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKYFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRENGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNHNAQALNTLVKQLSSKFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNETTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT 91 RNA sequence encoding a auguucguguuccuggugcugcugccucugguguccagccagugugugaaccugaucaccagaacaSARS-CoV-2 S protein from cagucauacaccaacagcuuuaccagaggcguguacuaccccgacaagguguucagauccagcgugan Omicron BA.4.6/BF.7 cugcacucuacccaggaccuguuccugccuuucuucagcaacgugaccugguuccacgccaucuccvariantggcaccaauggcaccaagagauucgacaaccccgugcugcccuucaacgacgggguguacuuugccagcaccgagaaguccaacaucaucagaggcuggaucuucggcaccacacuggacagcaagacccagagccugcugaucgugaacaacgccaccaacguggucaucaaagugugcgaguuccaguucugcaacgaccccuuccuggacgucuacuaccacaagaacaacaagagcuggauggaaagcgaguuccggguguacagcagcgccaacaacugcaccuucgaguacgugucccagccuuuccugauggaccuggaaggcaagcagggcaacuucaagaaccugcgcgaguucguguuuaagaacaucgacggcuacuucaagaucuacagcaagcacaccccuaucaaccucggccgggaucugccucagggcuucucugcucuggaaccccugguggaucugcccaucggcaucaacaucacccgguuucagacacugcuggcccugcacagaagcuaccugacaccuggcgauagcagcagcggauggacagcuggugccgccgcuuacuaugugggcuaccugcagccuagaaccuuccugcugaaguacaacgagaacggcaccaucaccgacgccguggauugugcucuggauccucugagcgagacaaagugcacccugaaguccuucaccguggaaaagggcaucuaccagaccagcaacuuccgggugcagcccaccgaauccaucgugcgguuccccaauaucaccaaucugugccccuucgacgagguguucaaugccaccaccuucgccucuguguacgccuggaaccggaagcggaucagcaauugcguggccgacuacuccgugcuguacaacuucgcccccuucuucgcauucaagugcuacggcguguccccuaccaagcugaacgaccugugcuucacaaacguguacgccgacagcuucgugauccggggaaacgaagugcggcagauugccccuggacagacaggcaacaucgccgacuacaacuacaagcugcccgacgacuucaccggcugugugauugccuggaacagcaacaagcuggacuccaaagucggcggcaacuacaauuacagguaccggcuguuccggaaguccaaucugaagcccuucgagcgggacaucuccaccgagaucuaucaggccggcaacaagccuuguaacggcguggcaggcgugaacugcuacuucccacugcaguccuacggcuuuaggcccacauacggcgugggccaccagcccuacagagugguggugcugagcuucgaacugcugcaugccccugccacagugugcggcccuaagaaaagcaccaaucucgugaagaacaaaugcgugaacuucaacuucaacggccugaccggcaccggcgugcugacagagagcaacaagaaguuccugccauuccagcaguuuggccgggauaucgccgauaccacagacgccguuagagauccccagacacuggaaauccuggacaucaccccuugcagcuucggcggagugucugugaucaccccuggcaccaacaccagcaaucagguggcagugcuguaccagggcgugaacuguaccgaagugcccguggccauucacgccgaucagcugacaccuacauggcggguguacuccaccggcagcaauguguuucagaccagagccggcugucugaucggagccgaguacgugaacaauagcuacgagugcgacauccccaucggcgcuggaaucugcgccagcuaccagacacagacaaagagccaccggagagccagaagcguggccagccagagcaucauugccuacacaaugucucugggcgccgagaacagcguggccuacuccaacaacucuaucgcuauccccaccaacuucaccaucagcgugaccacagagauccugccuguguccaugaccaagaccagcguggacugcaccauguacaucugcggcgauuccaccgagugcuccaaccugcugcugcaguacggcagcuucugcacccagcugaaaagagcccugacagggaucgccguggaacaggacaagaacacccaagagguguucgcccaagugaagcagaucuacaagaccccuccuaucaaguacuucggcggcuucaauuucagccagauucugcccgauccuagcaagcccagcaagcggagcuucaucgaggaccugcuguucaacaaagugacacuggccgacgccggcuucaucaagcaguauggcgauugucugggcgacauugccgccagggaucugauuugcgcccagaaguuuaacggacugacagugcugccuccucugcugaccgaugagaugaucgcccaguacacaucugcccugcuggccggcacaaucacaagcggcuggacauuuggagcaggcgccgcucugcagauccccuuugcuaugcagauggccuaccgguucaacggcaucggagugacccagaaugugcuguacgagaaccagaagcugaucgccaaccaguucaacagcgccaucggcaagauccaggacagccugagcagcacagcaagcgcccugggaaagcugcaggacguggucaaccacaaugcccaggcacugaacacccuggucaagcagcuguccuccaaguucggcgccaucagcucugugcugaacgauauccugagcagacuggacccuccugaggccgaggugcagaucgacagacugaucacaggcagacugcagagccuccagacauacgugacccagcagcugaucagagccgccgagauuagagccucugccaaucuggccgccaccaagaugucugagugugugcugggccagagcaagagaguggacuuuugcggcaagggcuaccaccugaugagcuucccucagucugccccucacggcgugguguuucugcacgugacauaugugcccgcucaagagaagaauuucaccaccgcuccagccaucugccacgacggcaaagcccacuuuccuagagaaggcguguucguguccaacggcacccauugguucgugacacagcggaacuucuacgagccccagaucaucaccaccgacaacaccuucgugucuggcaacugcgacgucgugaucggcauugugaacaauaccguguacgacccucugcagcccgagcuggacagcuucaaagaggaacuggacaaguacuuuaagaaccacacaagccccgacguggaccugggcgauaucagcggaaucaaugccagcgucgugaacauccagaaagagaucgaccggcugaacgagguggccaagaaucugaacgagagccugaucgaccugcaagaacuggggaaguacgagcaguacaucaaguggcccugguacaucuggcugggcuuuaucgccggacugauugccaucgugauggucacaaucaugcuguguugcaugaccagcugcuguagcugccugaagggcuguuguagcuguggcagcugcugcaaguucgacgaggacgauucugagcccgugcugaagggcgugaaacugcacuacacaugauga 92DNA sequence encoding a atgttcgtgttcctggtgctgctgcctctggtgtccagccagtgtgtgaacctgatcaccagaacaSARS-CoV-2 S protein from cagtcatacaccaacagctttaccagaggcgtgtactaccccgacaaggtgttcagatccagcgtgan Omicron BA.4.6/BF.7ctgcactctacccaggacctgttcctgcctttcttcagcaacgtgacctggttccacgccatctccvariantggcaccaatggcaccaagagattcgacaaccccgtgctgcccttcaacgacggggtgtactttgccagcaccgagaagtccaacatcatcagaggctggatcttcggcaccacactggacagcaagacccagagcctgctgatcgtgsacaacgccaccaacgtggtcatcaaagtgtgcgagttccagttctgcaacgaccccttcctggacgtctactaccacaagaacaacaagagctggatggaaagcgagttccgggtgtacagcagcgccaacaactgcaccttcgagtacgtgtcccagcctttcctgatggacctggaaggcaagcagggcaacttcaagaacctgcgcgagttcgtgtttaagaacatcgacggctacttcaagatctacagcaagcacacccctatcaacctcggccgggatctgcctcagggcttctctgctctggaacccctggtggatctgcccatcggcatcaacatcacccggtttcagacactgctggccctgcacagaagctacctgacacctggcgatagcagcagcggatggacagctggtgccgccgcttactatgtgggctacctgcagcctagaaccttcctgctgaagtacaacgagaacggcaccatcaccgacgccgtggattgtgctctggatcctctgagcgagacaaagtgcaccctgaagtccttcaccgtggaaaagggcatctaccagaccagcaacttccgggtgcagcccaccgaatccatcgtgcggttccccaatatcaccaatctgtgccccttcgacgaggtgttcaatgccaccaccttcgcctctgtgtacgcctggaaccggaagcggatcagcaattgcgtggccgactactccgtgctgtacaacttcgcccccttcttcgcattcaagtgctacggcgtgtcccctaccaagctgaacgacctgtgcttcacaaacgtgtacgccgacagcttcgtgatccggggaaacgaagtgcggcagattgcccctggacagacaggcaacatcgccgactacaactacaagctgcccgacgacttcaccggctgtgtgattgcctggaacagcaacaagctggactccaaagtcggcggcaactacaattacaggtaccggctgttccggaagtccaatctgaagcccttcgagcgggacatctccaccgagatctatcaggccggcaacaagccttgtaacggcgtggcaggcgtgaactgctacttcccactgcagtcctacggctttaggcccacatacggcgtgggccaccagccctacagagtggtggtgctgagcttcgaactgctgcatgcccctgccacagtgtgcggccctaagaaaagcaccaatctcgtgaagaacaaatgcgtgaacttcaacttcaacggcctgaccggcaccggcgtgctgacagagagcaacaagaagttcctgccattccagcagtttggccgggatatcgccgataccacagacgccgttagagatccccagacactggaaatcctggacatcaccccttgcagcttcggcggagtgtctgtgatcacccctggcaccaacaccagcaatcaggtggcagtgctgtaccagggcgtgaactgtaccgaagtgcccgtggccattcacgccgatcagctgacacctacatggcgggtgtactccaccggcagcaatgtgtttcagaccagagccggctgtctgatcggagccgagtacgtgaacaatagctacgagtgcgacatccccatcggcgctggaatctgcgccagctaccagacacagacaaagagccaccggagagccagaagcgtggccagccagagcatcattgcctacacaatgtctctgggcgccgagaacagcgtggcctactccaacaactctatcgctatccccaccaacttcaccatcagcgtgaccacagagatcctgcctgtgtccatgaccaagaccagcgtggactgcaccatgtacatctgcggcgattccaccgagtgctccaacctgctgctgcagtacggcagcttctgcacccagctgassagagccctgacagggatcgccgtggaacaggacaagaacacccaagaggtgttcgcccaagtgaagcagatctacaagacccctcctatcaagtacttcggcggcttcaatttcagccagattctgcccgatcctagcaagcccagcaagcggagcttcatcgaggacctgctgttcaacaaagtgacactggccgacgccggcttcatcaagcagtatggcgattgtctgggcgacattgccgccagggatctgatttgcgcccagaagtttaacggactgacagtgctgcctcctctgctgaccgatgagatgatcgcccagtacacatctgccctgctggccggcacaatcacaagcggctggacatttggagcaggcgccgctctgcagatcccctttgctatgcagatggcctaccggttcaacggcatcggagtgacccagaatgtgctgtacgagaaccagaagctgatcgccaaccagttcaacagcgccatcggcaagatccaggacagcctgagcagcacagcaagcgccctgggaaagctgcaggacgtggtcaaccacaatgcccaggcactgaacaccctggtcaagcagctgtcctccaagttcggcgccatcagctctgtgctgaacgatatcctgagcagactggaccctcctgaggccgaggtgcagatcgacagactgatcacaggcagactgcagagcctccagacatacgtgacccagcagctgatcagagccgccgagattagagcctctgccaatctggccgccaccaagatgtctgagtgtgtgctgggccagagcaagagagtggacttttgcggcaagggctaccacctgatgagcttccctcagtctgcccctcacggcgtggtgtttctgcacgtgacatatgtgcccgctcaagagaagaatttcaccaccgctccagccatctgccacgacggcaaagcccactttcctagagaaggcgtgttcgtgtccaacggcacccattggttcgtgacacagcggaacttctacgagccccagatcatcaccaccgacaacaccttcgtgtctggcaactgcgacgtcgtgatcggcattgtgaacaataccgtgtacgaccctctgcagcccgagctggacagcttcaaagaggaactggacaagtactttaagaaccacacaagccccgacgtggacctgggcgatatcagcggaatcaatgccagcgtcgtgaacatccagaaagagatcgaccggctgaacgaggtggccaagaatctgaacgagagcctgatcgacctgcaagaactggggaagtacgagcagtacatcaagtggccctggtacatctggctgggctttatcgccggactgattgccatcgtgatggtcacaatcatgctgtgttgcatgaccagctgctgtagctgcctgaagggctgttgtagctgtggcagctgctgcaagttcgacgaggacgattctgagcccgtgctgaagggcgtgaaactgcactacacatgatga 93Full length RNA constructagaauaaacuaguauucuucugguccccacagacucagagagaacccgccaccauguucguguuccencoding a SARS-COV-2 Suggugcugcugccucugguguccagccagugugugaaccugaucaccagaacacagucauacaccaprotein from an OmicronacagcuuuaccagaggcguguacuaccccgacaagguguucagauccagcgugcugcacucuacccBA.4.6/BF.7 variantaggaccuguuccugccuuucuucagcaacgugaccugguuccacgccaucuccggcaccaauggcaccaagagauucgacaaccccgugcugcccuucaacgacgggguguacuuugccagcaccgagaaguccaacaucaucagaggcuggaucuucggcaccacacuggacagcaagacccagagccugcugaucgugaacaacgccaccaacquggucaucaaagugugcgaguuccaguucugcaacgaccccuuccuggacgucuacuaccacaagaacaacaagagcuggauggaaagcgaguuccggguguacagcagcgccaacaacugcaccuucgaguacgugucccagccuuuccugauggaccuggaaggcaagcagggcaacuucaagaaccugcgcgaguucguguuuaagaacaucgacggcuacuucaagaucuacagcaagcacaccccuaucaaccucggccgggaucugccucagggcuucucugcucuggaaccccugguggaucugcccaucggcaucaacaucacccgguuucagacacugcuggcccugcacagaagcuaccugacaccuggcgauagcagcagcggauggacagcuggugccgccgcuuacuaugugggcuaccugcagccuagaaccuuccugcugaaguacaacgagaacggcaccaucaccgacgccguggauugugcucuggauccucugagcgagacaaagugcacccugaaguccuucaccguggaaaagggcaucuaccagaccagcaacuuccgggugcagcccaccgaauccaucgugcgguuccccaauaucaccaaucugugccccuucgacgagguguucaaugccaccaccuucgccucuguguacgccuggaaccggaagcggaucagcaauugcguggccgacuacuccgugcuguacaacuucgcccccuucuucgcauucaagugcuacggcguguccccuaccaagcugaacgaccugugcuucacaaacguguacgccgacagcuucgugauccggggaaacgaagugcggcagauugccccuggacagacaggcaacaucgccgacuacaacuacaagcugcccgacgacuucaccggcugugugauugccuggaacagcaacaagcuggacuccaaagucggcggcaacuacaauuacagguaccggcuguuccggaaguccaaucugaagcccuucgagcgggacaucuccaccgagaucuaucaggccggcaacaagccuuguaacggcguggcaggcgugaacugcuacuucccacugcaguccuacggcuuuaggcccacauacggcgugggccaccagcccuacagagugguggugcugagcuucgaacugcugcaugccccugccacagugugcggcccuaagaaaagcaccaaucucgugaagaacaaaugcgugaacuucaacuucaacggccugaccggcaccggcgugcugacagagagcaacaagaaguuccugccauuccagcaguuuggccgggauaucgccgauaccacagacgccguuagagauccccagacacuggaaauccuggacaucaccccuugcagcuucggcggagugucugugaucaccccuggcaccaacaccagcaaucagguggcagugcuguaccagggcgugaacuguaccgaagugcccguggccauucacgccgaucagcugacaccuacauggcggguguacuccaccggcagcaauguguuucagaccagagccggcugucugaucggagccgaguacgugaacaauagcuacgagugcgacauccccaucggcgcuggaaucugcgccagcuaccagacacagacaaagagccaccggagagccagaagcguggccagccagagcaucauugccuacacaaugucucugggcgccgagaacagcguggccuacuccaacaacucuaucgcuauccccaccaacuucaccaucagcgugaccacagagauccugccuguguccaugaccaagaccagcguggacugcaccauguacaucugcggcgauuccaccgagugcuccaaccugcugcugcaguacggcagcuucugcacccagcugaaaagagcccugacagggaucgccguggaacaggacaagaacacccaagagguguucgcccaagugaagcagaucuacaagaccccuccuaucaaguacuucggcggcuucaauuucagccagauucugcccgauccuagcaagcccagcaagcggagcuucaucgaggaccugcuguucaacaaagugacacuggccgacgccggcuucaucaagcaguauggcgauugucugggcgacauugccgccagggaucugauuugcgcccagaaguuuaacggacugacagugcugccuccucugcugaccgaugagaugaucgcccaguacacaucugcccugcuggccggcacaaucacaagcggcuggacauuuggagcaggcgccgcucugcagauccccuuugcuaugcagauggccuaccgguucaacggcaucggagugacccagaaugugcuguacgagaaccagaagcugaucgccaaccaguucaacagcgccaucggcaagauccaggacagccugagcagcacagcaagcgcccugggaaagcugcaggacguggucaaccacaaugcccaggcacugaacacccuggucaagcagcuguccuccaaguucggcgccaucagcucugugcugaacgauauccugagcagacuggacccuccugaggccgaggugcagaucgacagacugaucacaggcagacugcagagccuccagacauacgugacccagcagcugaucagagccgccgagauuagagccucugccaaucuggccgccaccaagaugucugagugugugcugggccagagcaagagaguggacuuuugcggcaagggcuaccaccugaugagcuucccucagucugccccucacggcgugguguuucugcacgugacauaugugcccgcucaagagaagaauuucaccaccgcuccagccaucugccacgacggcaaagcccacuuuccuagagaaggcguguucguguccaacggcacccauugguucgugacacagcggaacuucuacgagccccagaucaucaccaccgacaacaccuucgugucuggcaacugcgacgucgugaucggcauugugaacaauaccguguacgacccucugcagcccgagcuggacagcuucaaagaggaacuggacaaguacuuuaagaaccacacaagccccgacguggaccugggcgauaucagcggaaucaaugccagcgucgugaacauccagaaagagaucgaccggcugaacgagguggccaagaaucugaacgagagccugaucgaccugcaagaacuggggaaguacgagcaguacaucaaguggcccugguacaucuggcugggcuuuaucgccggacugauugccaucgugauggucacaaucaugcuguguugcaugaccagcugcuguagcugccugaagggcuguuguagcuguggcagcugcugcaaguucgacgaggacgauucugagcccgugcugaagggcgugaaacugcacuacacaugaugacucgagcugguacugcaugcacgcaaugcuagcugccccuuucccguccuggguaccccgagucucccccgaccucgggucccagguaugcucccaccuccaccugccccacucaccaccucugcuaguuccagacaccucccaagcacgcagcaaugcagcucaaaacgcuuagccuagccacacccccacgggaaacagcagugauuaaccuuuagcaauaaacgaaaguuuaacuaagcuauacuaaccccaggguuggucaauuucgugccagccacacccuggagcuagcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagcauaugacuaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 94 Full length DNA constructagaataaactagtattcttctggtccccacagactcagagagaacccgccaccatqttcgtgttccencoding a SARS-COV-2 Stggtgctgctgcctctggtgtccagccagtqtqtgaacctgatcaccagaacacagtcatacaccaprotein from an OmicronacagctttaccagaggcgtgtactaccccgacaaggtgttcagatccagcgtgctgcactctacccBA.4.6/BF.7 variantaggacctgttcctgcctttcttcagcaacgtgacctggttccacgccatctccggcaccaatggcaccaagagattcgacaaccccgtgctgcccttcaacgacggggtgtactttgccagcaccgagaagtccaacatcatcagaggctggatcttcggcaccacactggacagcaagacccagagcctgctgatcgtgaacaacgccaccaacgtggtcatcaaagtgtgcgagttccagttctgcaacgaccccttcctggacgtctactaccacaagaacaacaagagctggatggaaagcgagttccgggtgtacagcagcgccaacaactgcaccttcgagtacgtgtcccagcctttcctgatggacctggaaggcaagcagggcaacttcaagaacctgcgcgagttcgtgtttaagaacatcgacggctacttcaagatctacagcaagcacacccctatcaacctcggccgggatctgcctcagggcttctctgctctggaacccctggtggatctgcccatcggcatcaacatcacccggtttcagacactgctggccctgcacagaagctacctgacacctggcgatagcagcagcggatggacagctggtgccgccgcttactatgtgggctacctgcagcctagaaccttcctgctgaagtacaacgagaacggcaccatcaccgacgccgtggattgtgctctggatcctctgagcgagacaaagtgcaccctgaagtccttcaccgtggaaaagggcatctaccagaccagcaacttccgggtgcagcccaccgaatccatcgtgcggttccccaatatcaccaatctgtgccccttcgacgaggtgttcaatgccaccaccttcgcctctgtgtacgcctggaaccggaagcggatcagcaattgcgtggccgactactccgtgctgtacaacttcgcccccttcttcgcattcaagtgctacggcgtgtcccctaccaagctgaacgacctgtgcttcacaaacgtgtacgccgacagcttcgtgatccggggaaacgaagtgcggcagattgcccctggacagacaggcaacatcgccgactacaactacaagctgcccgacgacttcaccggctgtgtgattgcctggaacagcaacaagctggactccaaagtcggcggcaactacaattacaggtaccggctgttccggaagtccaatctgaagcccttcgagcgggacatctccaccgagatctatcaggccggcaacaagccttgtaacggcgtggcaggcgtgaactgctacttcccactgcagtcctacggctttaggcccacatacggcgtgggccaccagccctacagagtggtggtgctgagcttcgaactgctgcatgcccctgccacagtgtgcggccctaagaaaagcaccaatctcgtgaagaacaaatgcgtgaacttcaacttcaacggcctgaccggcaccggcgtgctgacagagagcaacaagaagttcctgccattccagcagtttggccgggatatcgccgataccacagacgccgttagagatccccagacactggaaatcctggacatcaccccttgcagcttcggcggagtgtctgtgatcacccctggcaccaacaccagcaatcaggtggcagtgctgtaccagggcgtgaactgtaccgaagtgcccgtggccattcacgccgatcagctgacacctacatggcgggtgtactccaccggcagcaatgtgtttcagaccagagccggctgtctgatcggagccgagtacgtgaacaatagctacgagtgcgacatccccatcggcgctggaatctgcgccagctaccagacacagacaaagagccaccggagagccagaagcgtggccagccagagcatcattgcctacacaatgtctctgggcgccgagaacagcgtggcctactccaacaactctatcgctatccccaccaacttcaccatcagcgtgaccacagagatcctgcctgtgtccatgaccaagaccagcgtggactgcaccatgtacatctgcggcgattccaccgagtgctccaacctgctgctgcagtacggcagcttctgcacccagctgaaaagagccctgacagggatcgccgtggaacaggacaagaacacccaagaggtgttcgcccaagtgaagcagatctacaagacccctcctatcaagtacttcggcggcttcaatttcagccagattctgcccgatcctagcaagcccagcaagcggagcttcatcgaggacctgctgttcaacaaagtgacactggccgacgccggcttcatcaagcagtatggcgattgtctgggcgacattgccgccagggatctgatttgcgcccagaagtttaacggactgacagtgctgcctcctctgctgaccgatgagatgatcgcccagtacacatctgccctgctggccggcacaatcacaagcggctggacatttggagcaggcgccgctctgcagatcccctttgctatgcagatggcctaccggttcaacggcatcggagtgacccagaatgtgctgtacgagaaccagaagctgatcgccaaccagttcaacagcgccatcggcaagatccaggacagcctgagcagcacagcaagcgccctgggaaagctgcaggacgtggtcaaccacaatgcccaggcactgaacaccctggtcaagcagctqtcctccaagttcggcgccatcagctctgtgctgaacgatatcctgagcagactggaccctcctgaggccgaggtgcagatcgacagactgatcacaggcagactgcagagcctccagacatacgtgacccagcagctgatcagagccgccgagattagagcctctgccaatctggccgccaccaagatgtctgagtgtgtgctgggccagagcaagagagtggacttttgcggcaagggctaccacctgatgagcttccctcagtctgcccctcacggcgtggtgtttctgcacgtgacatatgtgcccgctcaagagaagaatttcaccaccgctccagccatctgccacgacggcaaagcccactttcctagagaaggcgtgttcgtgtccaacggcacccattggttcgtgacacagcggaacttctacgagccccagatcatcaccaccgacaacaccttcgtgtctggcaactgcgacgtcgtgatcggcattgtgaacaataccgtgtacgaccctctgcagcccgagctggacagcttcaaagaggaactggacaagtactttaagaaccacacaagccccgacgtggacctgggcgatatcagcggaatcaatgccagcgtcgtgaacatccagaaagagatcgaccggctgaacgaggtggccaagaatctgaacgagagcctgatcgacctgcaagaactggggaagtacgagcagtacatcaagtggccctggtacatctggctgggctttatcgccggactgattgccatcgtgatggtcacaatcatgctgtgttgcatgaccagctgctgtagctgcctgaagggctgttgtagctgtggcagctgctgcaagttcgacgaggacgattctgagcccgctccacctgccccactcaccacctctgctagttccagacacctcccaagcacgcagcaatgcagcttgctgaagggcgtgaaactgcactacacatgatgactcgagctggtactgcatgcacgcaatgctagctgcccctttcccgtcctgggtaccccgagtctcccccgacctcgggtcccaggtatgctcccacctccacctgccccactcaccacctctgctagttccagacacctcccaagcacgcagcaatgcagctcaaaacgcttagcctagccacacccccacgggaaacagcagtgattaacctttagcaataaacgaaagtttaactaagctatactaaccccagggttggtcaatttcgtgccagccacaccctggagctagcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagcatatgactaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa

TABLE 15Sequence of one embodiment of an exemplary Omicron XBB-specific RNA vaccineSEQ ID NO. Brief Description Sequence 95 Amino acid sequence of RNA-MFVFLVLLPLVSSQCVNLITRTQSYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHencoded SARS-Cov-2 S proteinVSGTNGTKRFDNPALPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFfrom an Omicron XBB variantCNDPFLDVYQKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKEGNFKNLREFVFKNIDGYFK(with PRO mutations atIYSKHTPINLERDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGpositions corresponding toYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNK986P and V987P of SEQ ID LCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFNO: 1; i.e., PRO mutations VIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERat positions 981 and 982 of DISTEIYQAGNKPCNGVAGSNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNSEQ ID NO: 95)LVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEYVNNSYECDIPIGAGICASYQTQTKSHRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLKRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKYFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKENGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNHNAQALNTLVKQLSSKFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT 96 RNA sequence encoding aauguucguguuccuggugcugcugccucugguguccagccagugugugaaccugaucaccagaacaSARS-COV-2 S protein from cagUCAuacaccaacagouuuaccagaggcguguacuaccccgacaagguguucagauccagcgugan Omicron XBB variantcugcacucuacccaggaccuguuccugccuuucuucagcaacgugaccugguuccacgccauccacguguccggcaccaauggcaccaagagauucgacaaccccgcccugcccuucaacgacgggguguacuuugccagcaccgagaaguccaacaucaucagaggcuggaucuucggcaccacacuggacagcaagacccagagccugcugaucgugaacaacgccaccaacguggucaucaaagugugcgaguuccaguucugcaacgaccccuuccuggacgucuaccagaagaacaacaagagcuggauggaaagcgaguuccggguguacagcagcgccaacaacugcaccuucgaguacgugucccagccuuuccugauggaccuggaaggcaaggagggcaacuucaagaaccugcgcgaguucguguuuaagaacaucgacggcuacuucaagaucuacagcaagcacaccccuaucaaccucgagcgggaucugccucagggcuucucugcucuggaaccccugguggaucugcccaucggcaucaacaucacccgguuucagacacugcuggcccugcacagaagcuaccugacaccuggcgauagcagcagcggauggacagouggugccgccgcuuacuaugugggcuaccugcagccuagaaccuuccugcugaaguacaacgagaacggcaccaucaccgacgccguggauugugcucuggauccucugagcgagacaaagugcacccugaaguccuucaccguggaaaagggcaucuaccagaccagcaacuuccgggugcagcccaccgaauccaucgugcgguuccccaauaucaccaaucugugccccuuccacgagguguucaaugccaccaccuucgccucuguguacgccuggaaccggaagcggaucagcaauugcguggccgacuacuccgugaucuacaacuucgcccccuucuucgcauucaagugcuacggcguguccccuaccaagcugaacgaccugugcuucacaaacguguacgccgacagcuucgugauccggggaaacgaagugucacagauugccccuggacagacaggcaacaucgccgacuacaacuacaagcugcccgacgacuucaccgocugugugauugccuggaacagcaacaagcuggacuccaaacccagcggcaacuacaauuaccuguaccggcuguuccggaaguccaagcugaagcccuucgagcgggacaucuccaccgagaucuaucaggccggcaacaagccuuguaacggcguggcaggcagcaacugcuacagcccacugcaguccuacggcuuuaggcccacauacggcgugggccaccagcccuacagagugguggugcugagcuucgaacugcugcaugccccugccacagugugcggcccuaagassagcaccaaucucgugaagaacaaaugcgugaacuucaacuucaacggccugaccggcaccggcgugcugacagagagcaacaagaaguuccugccauuccagcaguuuggccgggauaucgccgauaccacagacgccguuagagauccccagacacuggaaauccuggacaucaccccuugcagcuucggcggagugucugugaucaccccuggcaccaacaccagcaaucagguggcagugcuguaccagggcgugaacuguaccgaagugcccguggccauucacgccgaucagcugacaccuacauggcggguguacuccaccggcagcaauguguuucagaccagagcoggcugucugaucggagccgaguacgugaacaauagcuacgagugcgacauccccaucggcgcuggaaucugcgccagcuaccagacacagacaaagagccaccggagagccagaagcguggccagccagagcaucauugccuacacaaugucucugggcgccgagaacagcguggccuacuccaacaacucuaucgcuauccccaccaacuucaccaucagcgugaccacagagauccugccuguguccaugaccaagaccagoguggacugcaccauguacaucugcggcgauuccaccgagugcuccaaccugcugcugcaguacggcagouucugcacccagcugaaaagagcccugacagggaucgccguggaacaggacaagaacacccaagagguguucgcccaagugaagcagaucuacaagaccccuccuaucaaguacuucggcggcuucaauuucagccagauucugcccgauccuagcaagcccagcaagcggagcuucaucgaggaccugcuguucaacaaagugacacuggccgacgccggcuucaucaagcaguauggcgauugucugggcgacauugccgccagggaucugauuugcgcccagaaguuuaacggacugacagugcugccuccucugcugaccgaugagaugaucgcccaguacacaucugcccugcuggccggcacaaucacaagcggcuggacauuuggagcaggcgccgcucugcagauccccuuugcuaugcagauggccuaccgguucaacggcaucggagugacccagaaugugcuguacgagaaccagaagcugaucgccaaccaguucaacagcgccaucggcaagauccaggacagccugagcagcacagcaagcgcccugggaaagcugcaggacguggucaaccacaaugcccaggcacugaacacccuggucaagcagcuguccuccaaguucggcgccaucagcucugugougaacgauauccugagcagacuggacccuccugaggccgaggugcagaucgacagacugaucacaggcagacugcagagccuccagacauacgugacccagcagcugaucagagccgccgagauuagagccucugccaaucuggccgccaccaagaugucugagugugugcugggccagagcaagagaguggacuuuugcggcaagggcuaccaccugaugagcuucccucagucugccccucacggcgugguguuucugcacgugacauaugugcccgcucaagagaagaauuucaccaccgcuccagccaucugccacgacggcaaagcccacuuuccuagagaaggcguguucguguccaacggcacccauugguucgugacacagcggaacuucuacgagccccagaucaucaccaccgacaacaccuucgugucuggcaacugcgacgucgugaucggcauugugaacaauaccguguacgacccucugcagcccgagcuggacagcuucaaagaggaacuggacaaguacuuuaagaaccacacaagccccgacguggaccugggcgauaucagcggaaucaaugccagcgucgugaacauccagaaagagaucgaccggcugaacgagguggccaagaaucugaacgagagccugaucgaccugcaagaacuggggaaguacgagcaguacaucaaguggcccugguacaucuggcugggcuuuaucgccggacugauugccaucgugauggucacaaucaugcuguguugcaugaccagougouguagcugccugaagggcuguuguagcuguggcagcugcugcaaguucgacgaggacgauucugagcccgugougaagggcgugaaacugcacuacacaugauga 97DNA sequence encoding aatgttcgtgttcctggtgctgctgcctctggtgtccagccagtgtgtgaacctgatcaccagaacaSARS-CoV-2 S protein fromcagTCAtacaccaacagctttaccagaggcgtgtactaccccgacaaggtgttcagatccagcgtgan Omicron XBB variantctgcactctacccaggacctgttcctgcctttcttcagcaacgtgacctggttccacgccatccacgtgtccggcaccaatggcaccaagagattcgacaaccccgccctgcccttcaacgacggggtgtactttgccagcaccgagaagtccaacatcatcagaggctggatcttcggcaccacactggacagcaagacccagagcctgctgatcgtgaacaacgccaccaacgtggtcatcaaagtgtgcgagttccagttctgcaacgaccccttcctggacgtctaccagaagaacaacaagagctggatggaaagcgagttccgggtgtacagcagcgccaacaactgcaccttcgagtacgtgtcccagcctttcctgatggacctggaaggcaaggagggcaacttcaagaacctgcgcgagttcgtgtttaagaacatcgacggctacttcaagatctacagcaagcacacccctatcaacctcgagcgggatctgcctcagggcttctctgctctggaacccctggtggatctgcccatcggcatcaacatcacccggtttcagacactgctggccctgcacagaagctacctgacacctggcgatagcagcagcggatggacagctggtgccgccgcttactatgtgggctacctgcagcctagaaccttcctgctgaagtacaacgagaacggcaccatcaccgacgccgtggattgtgctctggatcctctgagcgagacaaagtgcaccctgaagtccttcaccgtggaaaagggcatctaccagaccagcaacttccgggtgcagcccaccgaatccatcgtgcggttccccaatatcaccaatctgtgccccttccacgaggtgttcaatgccaccaccttcgcctctgtgtacgcctggaaccggaagcggatcagcaattgcgtggccgactactccgtgatctacaacttcgcccccttcttcgcattcaagtgctacggcgtgtcccctaccaagctgaacgacctgtgcttcacaaacgtgtacgccgacagcttcgtgatccggggaaacgaagtgtcacagattgcccctggacagacaggcaacatcgccgactacaactacaagctgcccgacgacttcaccggctgtgtgattgcctggaacagcaacaagctggactccaaacccagcggcaactacaattacctgtaccggctgttccggaagtccaagctgaagcccttcgagcgggacatctccaccgagatctatcaggccggcaacaagccttgtaacggcgtggcaggcagcaactgctacagcccactgcagtcctacggctttaggcccacatacggcgtgggccaccagccctacagagtggtggtgctgagcttcgaactgctgcatgcccctgccacagtgtgcggccctaagaaaagcaccaatctcgtgaagaacaaatgcgtgaacttcaacttcaacggcctgaccggcaccggcgtgctgacagagagcaacaagaagttcctgccattccagcagtttggccgggatatcgccgataccacagacgccgttagagatccccagacactggaaatcctggacatcaccccttgcagcttcggcggagtgtctgtgatcacccctggcaccaacaccagcaatcaggtggcagtgctgtaccagggcgtgaactgtaccgaagtgcccgtggccattcacgccgatcagctgacacctacatggcgggtgtactccaccggcagcaatgtgtttcagaccagagccggctgtctgatcggagccgagtacgtgaacaatagctacgagtgcgacatccccatcggcgctggaatctgcgccagctaccagacacagacaaagagccaccggagagccagaagcgtggccagccagagcatcattgcctacacaatgtctctgggcgccgagaacagcgtggcctactccaacaactctatcgctatccccaccaacttcaccatcagcgtgaccacagagatcctgcctgtgtccatgaccaagaccagcgtggactgcaccatgtacatctgcggcgattccaccgagtgctccaacctgctgctgcagtacggcagcttctgcacccagctgaaaagagccctgacagggatcgccgtggaacaggacaagaacacccaagaggtgttcgcccaagtgaagcagatctacaagacccctcctatcaagtacttcggcggcttcaatttcagccagattctgcccgatcctagcaagcccagcaagcggagcttcatcgaggacctgctgttcaacaaagtgacactggccgacgccggcttcatcaagcagtatggcgattgtctgggcgacattgccgccagggatctgatttgcgcccagaagtttaacggactgacagtgctgcctcctctgctgaccgatgagatgatcgcccagtacacatctgccctgctggccggcacaatcacaagcggctggacatttggagcaggcgccgctctgcagatcccctttgctatgcagatggcctaccggttcaacggcatcggagtgacccagaatgtgctgtacgagaaccagaagctgatcgccaaccagttcaacagcgccatcggcaagatccaggacagcctgagcagcacagcaagcgccctgggaaagctgcaggacgtggtcaaccacaatgcccaggcactgaacaccctggtcaagcagctgtcctccaagttcggcgccatcagctctgtgctgaacgatatcctgagcagactggaccctcctgaggccgaggtqcagatcgacagactgatcacaggcagactgcagagcctccagacatacgtgacccagcagctgatcagagccgccgagattagagcctctgccaatctggccgccaccaagatgtctgagtgtgtgctgggccagagcaagagagtggacttttgcggcaagggctaccacctgatgagcttccctcagtctgcccctcacggcgtggtgtttctgcacgtgacatatgtgcccgctcaagagaagaatttcaccaccgctccagccatctgccacgacggcaaagcccactttcctagagaaggcgtgttcgtgtccaacggcacccattggttcgtgacacagcggaacttctacgagccccagatcatcaccaccgacaacaccttcgtgtctggcaactgcgacgtcgtgatcggcattgtgaacaataccgtgtacgaccctctgcagcccgagctggacagcttcaaagaggaactggacaagtactttaagaaccacacaagccccgacgtggacctgggcgatatcagcggaatcaatgccagcgtcgtgaacatccagaaagagatcgaccggctgaacgaggtggccaagaatctgaacgagagcctgatcgacctgcaagaactggggaagtacgagcagtacatcaagtggccctggtacatctggctgggctttatcgccggactgattgccatcgtgatggtcacaatcatgctgtgttgcatgaccagctgctgtagctgcctgaagggctgttgtagctgtggcagctgctgcaagttcgacgaggacgattctgagcccgtgctgaagggcgtgaaactgcactacacatgatga 98Full length RNA constructagaauaaacuaguauucuucugguccccacagacucagagagaacccgccaccauguucguguuccencoding a SARS-COV-2 SuggugcugcugccucugguguccagccagugugugaaccugaucaccagaacacagUCAuacaccaprotein from an Omicron acagcuuuaccagaggcguguacuaccccgacaagguguucagauccagcgugcugcacucuacccXBB variantaggaccuguuccugccuuucuucagcaacgugaccugguuccacgccauccacguguccggcaccaauggcaccaagagauucgacaaccccgcccugcccuucaacgacgggguguacuuugccagcaccgagaaguccaacaucaucagaggcuggaucuucggcaccacacuggacagcaagacccagagccugcugaucgugaacaacgccaccaacguggucaucaaagugugcgaguuccaguucugcaacgaccccuuccuggacgucuaccagaagaacascaagagcuggauggaaagcgaguuccggguguacagcagcgccaacaacugcaccuucgaguacgugucccagccuuuccugauggaccuggaaggcaaggagggcaacuucaagaaccugcgcgaguucguguuuaagaacaucgacggcuacuucaagaucuacagcaagcacaccccuaucaaccucgagcgggaucugccucagggcuucucugcucuggaaccccugguggaucugcccaucggcaucaacaucacccgguuucagacacugcuggcccugcacagaagcuaccugacaccuggcgauagcagcagcggauggacagcuggugccgccgcuuacuaugugggcuaccugcagccuagaaccuuccugcugaaguacaacgagaacggcaccaucaccgacgccguggauugugcucuggauccucugagcgagacaaagugcacccugaaguccuucaccguggaaaagggcaucuaccagaccagcaacuuccgggugcagcccaccgaauccaucgugcgguuccccaauaucaccaaucugugccccuuccacgagguguucaaugccaccaccuucgccucuguguacgccuggaaccggaagcggaucagcaauugcguggccgacuacuccgugaucuacaacuucgcccccuucuucgcauucaagugcuacggcguguccccuaccaagcugaacgaccugugcuucacaaacguguacgccgacagcuucgugauccggggaaacgaagugucacagauugccccuggacagacaggcaacaucgccgacuacaacuacaagcugcccgacgacuucaccggcugugugauugccuggaacagcaacaagcuggacuccaaacccagcggcaacuacaauuaccuguaccggcuguuccggaaguccaagcugaagcccuucgagcgggacaucuccaccgagaucuaucaggccggcaacaagccuuguaacggcguggcaggcagcaacugcuacagcccacugcaguccuacggcuuuaggcccacauacggcgugggccaccagcccuacagagugguggugcugagcuucgaacugcugcaugccccugccacagugugcggcccuaagaaaagcaccaaucucgugaagaacaaaugcgugaacuucaacuucaacggccugaccggcaccggcgugcugacagagagcaacaagaaguuccugccauuccagcaguuuggccgggauaucgccgauaccacagacgccguuagagauccccagacacuggaaauccuggacaucaccccuugcagcuucggcggagugucugugaucaccccuggcaccaacaccagcaaucagguggcagugcuguaccagggcgugaacuguaccgaagugcccguggccauucacgccgaucagcugacaccuacauggcggguguacuccaccggcagcaauguguuucagaccagagccggcugucugaucggagccgaquacgugaacaauagcuacgagugcgacauccccaucggcgcuggaaucugcgccagcuaccagacacagacaaagagccaccggagagccagaagcguggccagccagagcaucauugccuacacaaugucucuggggcgcgagaacagcguggccuacuccaacaacucuaucgcuauccccaccaacuucaccaucagcgugaccacagagauccugccuguguccaugaccaagaccagcguggacugcaccauguacaucugcggcgauuccaccgagugcuccaaccugcugcugcaguacggcagcuucugcacccagcugaaaagagcccugacagggaucgccguggaacaggacaagaacacccaagagguguucgcccaagugaagcagaucuacaagaccccuccuaucaaguacuucggcggcuucaauuucagccagauucugcccgauccuagcaagcccagcaagcggagcuucaucgaggaccugcuguucaacaaagugacacuggccgacgccggcuucaucaagcaguauggcgauugucugggcgacauugccgccagggaucugauuugcgcccagaaguuuaacggacugacagugcugccuccucugcugaccgaugagaugaucgcccaguacacaucugcccugcuggccggcacaaucacaagcggcuggacauuuggagcaggcgccgcucugcagauccccuuugcuaugcagauggccuaccgguucaacggcaucggagugacccagaaugugcuguacgagaaccagaagcugaucgccaaccaguucaacagcgccaucggcaagauccaggacagccugagcagcacagcaagcgcccugggaaagcugcaggacguggucaaccacaaugcccaggcacugaacacccuggucaagcagcuguccuccaaguucggcgccaucagcucugugcugaacgauauccugagcagacuggacccuccugaggccgaggugcagaucgacagacugaucacaggcagacugcagagccuccagacauacgugacccagcagcugaucagagccgccgagauuagagccucugccaaucuggccgccaccaagaugucugagugugugcugggccagagcaagagaguggacuuuugcggcaagggcuaccaccugaugagcuucccucagucugccccucacggcgugguguuucugcacgugacauaugugcccgcucaagagaagaauuucaccaccgcuccagccaucugccacgacggcaaagcccacuuuccuagagaaggcguguucguguccaacggcacccauugguucgugacacagcggaacuucuacgagccccagaucaucaccaccgacaacaccuucgugucuggcaacugcgacgucgugaucggcauugugaacaauaccguguacgacccucugcagcccgagcuggacagcuucaaagaggaacuggacaaguacuuuaagaaccacacaagccccgacguggaccugggcgauaucagcggaaucaaugccagcgucgugaacauccagaaagagaucgaccggcugaacgagguggccaagaaucugaacgagagccugaucgaccugcaagaacuggggaaguacgagcaguacaucaaguggcccugguacaucuggcugggcuuuaucgccggacugauugccaucgugauggucacaaucaugcuguguugcaugaccagcugcuguagcugccugaagggcuguuguagcuguggcagcugcugcaaguucgacgaggacgauucugagcccgugcugaagggcgugaaacugcacuacacaugaugacucgagcugguacugcaugcacgcaaugcuagcugccccuuucccguccuggguaccccgagucucccccgaccucgggucccagguaugcucccaccuccaccugccccacucaccaccucugcuaguuccagacaccucccaagcacgcagcaaugcagcucaaaacgcuuagccuagccacacccccacgggaaacagcagugauuaaccuuuagcaauaaacgaaaguuuaacuaagcuauacuaaccccaggguuggucaauuucgugccagccacacccuggagcuagcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagcauaugacuaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 99Full length DNA constructagaataaactagtattcttctggtccccacagactcagagagaacccqccaccatgttcgtgttccencoding a SARS-COV-2 StggtgctgctgcctctggtgtccagccagtgtgtgaacctgatcaccagaacacagTCAtacaccaprotein from an Omicron acagctttaccagaggcgtgtactaccccgacaaggtgttcagatccagcgtgctgcactctacccXBB variantaggacctgttcctgcctttcttcagcaacgtgacctggttccacgccatccacgtgtccggcaccaatggcaccaagagattcgacaaccccgccctgcccttcaacgacggggtgtactttgccagcaccgagaagtccaacatcatcagaggctggatcttcggcaccacactggacagcaagacccagagcctgctgatcgtgaacaacgccaccaacgtggtcatcaaagtgtgcgagttccagttctgcaacgaccccttcctggacgtctaccagaagaacaacaagagctggatggaaagcgagttccgggtgtacagcagcgccaacaactgcaccttcgagtacgtgtcccagcctttcctgatggacctggaaggcaaggagggcaacttcaagaacctgcgcgagttcgtgtttaagaacatcgacggctacttcaagatctacagcaagcacacccctatcaacctcgagcgggatctgcctcagggcttctctgctctggaacccctggtggatctgcccatcggcatcaacatcacccggtttcagacactgctggccctgcacagaagctacctgacacctggcgatagcagcagcggatggacagctggtgccgccgcttactatgtgggctacctgcagcctagaaccttcctgctgaagtacaacgagaacggcaccatcaccgacgccgtggattgtgctctggatcctctgagcgagacasagtgcaccctgaagtccttcaccgtggaaaagggcatctaccagaccagcaacttccgggtgcagcccaccgaatccatcgtgcggttccccaatatcaccaatctgtgccccttccacgaggtgttcaatgccaccaccttcgcctctgtgtacgcctggaaccggaagcggatcagcaattgcgtggccgactactccgtgatctacaacttcgcccccttcttcgcattcaagtgctacggcgtgtcccctaccaagctgaacgacctgtgcttcacaaacgtgtacgccgacagcttcgtgatccggggaaacgaagtgtcacagattccccctggacagacaggcaacatcgccgactacaactacaagctgcccgacgacttcaccggctgtgtgattgcctggaacagcaacaagctggactccaaacccagcggcaactacaattacctgtaccggctgttccggaagtccaagctgaagcccttcgagcgggacatctccaccgagatctatcaggccggcaacaagccttgtaacggcgtggcaggcagcaactgctacagcccactgcagtcctacggctttaggcccacatacggcgtgggccaccagccctacagagtggtggtgctgagcttcgaactgctgcatgcccctgccacagtgtgcggccctaagaaaagcaccaatctcgtgaagaacaaatgcgtgaacttcaacttcaacggcctgaccggcaccggcgtgctgacagagagcaacaagaagttcctgccattccagcagtttggccgggatatcgccgataccacagacgccgttagagatccccagacactggaaatcctggacatcaccccttgcagcttcggcggagtgtctgtgatcacccctggcaccaacaccagcaatcaggtcgcagtgctgtaccagggcgtgaactgtaccgaagtgcccgtggccattcacgccgatcagctgacacctacatggcgggtgtactccaccggcagcaatgtgtttcagaccagagccggctgtctgatcggagccgagtacgtgaacaatagctacgagtgcgacatccccatcggcgctggaatctgcgccagctaccagacacagacaaagagccaccggagagccagaagcgtggccagccagagcatcattgcctacacaatgtctctgggcgccgagaacagcgtggcctactccaacaactctatcgctatccccaccaacttcaccatcagcgtgaccacagagatcctgcctgtgtccatgaccaagaccagcgtggactgcaccatgtacatctgcggcgattccaccgagtgctccaacctgctgctgcagtacggcagcttctgcacccagctgaaaagagccctgacagggatcgccgtggaacaggacaagaacacccaagaggtgttcgcccaagtgaagcagatctacaagacccctcctatcaagtacttcggcggcttcaatttcagccagattctgcccgatcctagcaagcccagcaagcggagcttcatcgaggacctgctgttcaacaaagtgacactggccgacgccggcttcatcaagcagtatggcgattgtctgggcgacattgccgccagggatctgatttccgcccagaagtttaacggactgacagtgctgcctcctctgctgaccgatgagatgatcgcccagtacacatctgccctgctggccggcacaatcacaagcggctggacatttggagcaggcgccgctctgcagatcccctttgctatgcagatggcctaccggttcaacggcatcggagtgacccagaatgtgctgtacgagaaccagaagctgatcgccaaccagttcaacagcgccatcggcaagatccaggacagcctgagcagcacagcaagcgccctgggaaagctgcaggacgtggtcaaccacaatgcccaggcactgaacaccctggtcaagcagctgtcctccaagttcggcgccatcagctctgtgctgaacgatatcctgagcagactggaccctcctgaggccgaggtgcagatcgacagactgatcacaggcagactgcagagcctccagacatacgtgacccagcagctgatcagagccgccgagattagagcctctgccaatctggccgccaccaagatgtctgagtgtgtgctgggccagagcaagagagtggacttttgcggcaagggctaccacctgatgagcttccctcagtctgcccctcacggcgtggtgtttctgcacgtgacatatgtgcccgctcaagagaagaatttcaccaccgctccagccatctgccacgacggcaaagcccactttcctagagaaggcgtgttcgtgtccaacggcacccattggttcgtgacacagcggaacttctacgagccccagatcatcaccaccgacaacaccttcgtgtctggcaactgcgacgtcgtgatcggcattgtgaacaataccgtgtacgaccctctgcagcccgagctggacagcttcaaagaggaactggacaagtactttaagaaccacacaagccccgacgtggacctgggcgatatcagcggaatcaatgccagcgtcgtgaacatccagaaagagatcgaccggctgaacgaggtggccaagaatctgaacgagagcctgatcgacctgcaagaactggggsagtacgagcagtacatcaagtggccctggtacatctggctgggctttatcgccggactgattgccatcgtgatggtcacaatcatgctgtgttgcatgaccagctgctgtagctgcctgaagggctgttgtagctgtggcagctgctgcaagttcgacgaggacgattctgagcccgcgctgaagggcgtgaaactgcactacacatgatgactcgagctggtactgcatgcacgcaatgctagctgcccctttcccgtcctgggtaccccgagtctcccccgacctcgggtcccaggtatgctcccacctccacctgccccactcaccacctctgctagttccagacacctcccaagcacgcagcaatgcagctcaaaacgcttagcctagccacacccccacgggaaacagcagtgattaacctttagcaataaacgaaagtttaactaagctatactaaccccagggttggtcaatttcgtgccagccacaccctggagctagcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagcatatgactaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa

TABLE 16Sequence of one embodiment of an exemplary Omicron BQ.1.1-specific RNA vaccineSEQ ID NO. Brief Description Sequence 100 Amino acid sequence of MFVFLVLLPLVSSQCVNLITRTQSYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAISRNA-encoded SARS-CoV-2 SGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNprotein from an Omicron DPFLDVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIBQ.1.1 variant (with PRO muta- YSKHTPINLGRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYtions at positions correspond-LQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLing to K986P and V987P of CPFDEVENATTFASVYAWNRKRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVSEQ ID NO: 1; i.e., PROIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSTVGGNYNYRYRLFRKSKLKPFERDmutations at positions 981ISTEIYQAGNKPCNGVAGVNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLand 982 of SEQ ID NO: 100)VKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEYVNNSYECDIPIGAGICASYQTQTKSHRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLKRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKYF  GGFNFSQILPDPSKPSKRSFIEDLLENKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRENGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNHNAQALNTLVKQLSSKFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNETTAPAICHDGKAHFPREGVEVSNGTHWEVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT 101 RNA sequence encoding aAUGUUCGUGUUCCUGGUGCUGCUGCCUCUGGUGUCCAGCCAGUGUGUGAACCUGAUCACCAGAACASARS-COV-2 S protein from CAGUCAUACACCAACAGCUUUACCAGAGGCGUGUACUACCCCGACAAGGUGUUCAGAUCCAGCGUGan Omicron BQ.1.1 variantCUGCACUCUACCCAGGACCUGUUCCUGCCUUUCUUCAGCAACGUGACCUGGUUCCACGCCAUCUCCGGCACCAAUGGCACCAAGAGAUUCGACAACCCCGUGCUGCCCUUCAACGACGGGGUGUACUUUGCCAGCACCGAGAAGUCCAACAUCAUCAGAGGCUGGAUCUUCGGCACCACACUGGACAGCAAGACCCAGAGCCUGCUGAUCGUGAACAACGCCACCAACGUGGUCAUCAAAGUGUGCGAGUUCCAGUUCUGCAACGACCCCUUCCUGGACGUCUACUACCACAAGAACAACAAGAGCUGGAUGGAAAGCGAGUUCCGGGUGUACAGCAGCGCCAACAACUGCACCUUCGAGUACGUGUCCCAGCCUUUCCUGAUGGACCUGGAAGGCAAGCAGGGCAACUUCAAGAACCUGCGCGAGUUCGUGUUUAAGAACAUCGACGGCUACUUCAAGAUCUACAGCAAGCACACCCCUAUCAACCUCGGCCGGGAUCUGCCUCAGGGCUUCUCUGCUCUGGAACCCCUGGUGGAUCUGCCCAUCGGCAUCAACAUCACCCGGUUUCAGACACUGCUGGCCCUGCACAGAAGCUACCUGACACCUGGCGAUAGCAGCAGCGGAUGGACAGCUGGUGCCGCCGCUUACUAUGUGGGCUACCUGCAGCCUAGAACCUUCCUGCUGAAGUACAACGAGAACGGCACCAUCACCGACGCCGUGGAUUGUGCUCUGGAUCCUCUGAGCGAGACAAAGUGCACCCUGAAGUCCUUCACCGUGGAAAAGGGCAUCUACCAGACCAGCAACUUCCGGGUGCAGCCCACCGAAUCCAUCGUGCGGUUCCCCAAUAUCACCAAUCUGUGCCCCUUCGACGAGGUGUUCAAUGCCACCACCUUCGCCUCUGUGUACGCCUGGAACCGGAAGCGGAUCAGCAAUUGCGUGGCCGACUACUCCGUGCUGUACAACUUCGCCCCCUUCUUCGCAUUCAAGUGCUACGGCGUGUCCCCUACCAAGCUGAACGACCUGUGCUUCACAAACGUGUACGCCGACAGCUUCGUGAUCCGGGGAAACGAAGUGUCACAGAUUGCCCCUGGACAGACAGGCAACAUCGCCGACUACAACUACAAGCUGCCCGACGACUUCACCGGCUGUGUGAUUGCCUGGAACAGCAACAAGCUGGACUCCACCGUCGGCGGCAACUACAAUUACAGGUACCGGCUGUUCCGGAAGUCCAAGCUGAAGCCCUUCGAGCGGGACAUCUCCACCGAGAUCUAUCAGGCCGGCAACAAGCCUUGUAACGGCGUGGCAGGCGUGAACUGCUACUUCCCACUGCAGUCCUACGGCCUUAGGCCCACAUACGGCGUGGGCCACCAGCCCUACAGAGUGGUGGUGCUGAGCUUCGAACUGCUGCAUGCCCCUGCCACAGUGUGCGGCCCUAAGAAAAGCACCAAUCUCGUGAAGAACAAAUGCGUGAACUUCAACUUCAACGGCCUGACCGGCACCGGCGUGCUGACAGAGAGCAACAAGAAGUUCCUGCCAUUCCAGCAGUUUGGCCGGGAUAUCGCCGAUACCACAGACGCCGUUAGAGAUCCCCAGACACUGGAAAUCCUGGACAUCACCCCUUGCAGCUUCGGCGGAGUGUCUGUGAUCACCCCUGGCACCAACACCAGCAAUCAGGUGGCAGUGCUGUACCAGGGCGUGAACUGUACCGAAGUGCCCGUGGCCAUUCACGCCGAUCAGCUGACACCUACAUGGCGGGUGUACUCCACCGGCAGCAAUGUGUUUCAGACCAGAGCCGGCUGUCUGAUCGGAGCCGAGUACGUGAACAAUAGCUACGAGUGCGACAUCCCCAUCGGCGCUGGAAUCUGCGCCAGCUACCAGACACAGACAAAGAGCCACCGGAGAGCCAGAAGCGUGGCCAGCCAGAGCAUCAUUGCCUACACAAUGUCUCUGGGCGCCGAGAACAGCGUGGCCUACUCCAACAACUCUAUCGCUAUCCCCACCAACUUCACCAUCAGCGUGACCACAGAGAUCCUGCCUGUGUCCAUGACCAAGACCAGCGUGGACUGCACCAUGUACAUCUGCGGCGAUUCCACCGAGUGCUCCAACCUGCUGCUGCAGUACGGCAGCUUCUGCACCCAGCUGAAAAGAGCCCUGACAGGGAUCGCCGUGGAACAGGACAAGAACACCCAAGAGGUGUUCGCCCAAGUGAAGCAGAUCUACAAGACCCCUCCUAUCAAGUACUUCGGCGGCUUCAAUUUCAGCCAGAUUCUGCCCGAUCCUAGCAAGCCCAGCAAGCGGAGCUUCAUCGAGGACCUGCUGUUCAACAAAGUGACACUGGCCGACGCCGGCUUCAUCAAGCAGUAUGGCGAUUGUCUGGGCGACAUUGCCGCCAGGGAUCUGAUUUGCGCCCAGAAGUUUAACGGACUGACAGUGCUGCCUCCUCUGCUGACCGAUGAGAUGAUCGCCCAGUACACAUCUGCCCUGCUGGCCGGCACAAUCACAAGCGGCUGGACAUUUGGAGCAGGCGCCGCUCUGCAGAUCCCCUUUGCUAUGCAGAUGGCCUACCGGUUCAACGGCAUCGGAGUGACCCAGAAUGUGCUGUACGAGAACCAGAAGCUGAUCGCCAACCAGUUCAACAGCGCCAUCGGCAAGAUCCAGGACAGCCUGAGCAGCACAGCAAGCGCCCUGGGAAAGCUGCAGGACGUGGUCAACCACAAUGCCCAGGCACUGAACACCCUGGUCAAGCAGCUGUCCUCCAAGUUCGGCGCCAUCAGCUCUGUGCUGAACGAUAUCCUGAGCAGACUGGACCCUCCUGAGGCCGAGGUGCAGAUCGACAGACUGAUCACAGGCAGACUGCAGAGCCUCCAGACAUACGUGACCCAGCAGCUGAUCAGAGCCGCCGAGAUUAGAGCCUCUGCCAAUCUGGCCGCCACCAAGAUGUCUGAGUGUGUGCUGGGCCAGAGCAAGAGAGUGGACUUUUGCGGCAAGGGCUACCACCUGAUGAGCUUCCCUCAGUCUGCCCCUCACGGCGUGGUGUUUCUGCACGUGACAUAUGUGCCCGCUCAAGAGAAGAAUUUCACCACCGCUCCAGCCAUCUGCCACGACGGCAAAGCCCACUUUCCUAGAGAAGGCGUGUUCGUGUCCAACGGCACCCAUUGGUUCGUGACACAGCGGAACUUCUACGAGCCCCAGAUCAUCACCACCGACAACACCUUCGUGUCUGGCAACUGCGACGUCGUGAUCGGCAUUGUGAACAAUACCGUGUACGACCCUCUGCAGCCCGAGCUGGACAGCUUCAAAGAGGAACUGGACAAGUACUUUAAGAACCACACAAGCCCCGACGUGGACCUGGGCGAUAUCAGCGGAAUCAAUGCCAGCGUCGUGAACAUCCAGAAAGAGAUCGACCGGCUGAACGAGGUGGCCAAGAAUCUGAACGAGAGCCUGAUCGACCUGCAAGAACUGGGGAAGUACGAGCAGUACAUCAAGUGGCCCUGGUACAUCUGGCUGGGCUUUAUCGCCGGACUGAUUGCCAUCGUGAUGGUCACAAUCAUGCUGUGUUGCAUGACCAGCUGCUGUAGCUGCCUGAAGGGCUGUUGUAGCUGUGGCAGCUGCUGCAAGUUCGACGAGGACGAUUCUGAGCCCGUGCUGAAGGGCGUGAAACUGCACUACACAUGAUGA 102DNA sequence encoding a ATGTTCGTGTTCCTGGTGCTGCTGCCTCTGGTGTCCAGCCAGTGTGTGAACCTGATCACCAGAACASARS-CoV-2 S protein from CAGTCATACACCAACAGCTTTACCAGAGGCGTGTACTACCCCGACAAGGTGTTCAGATCCAGCGTGan Omicron BQ.1.1 variantCTGCACTCTACCCAGGACCTGTTCCTGCCTTTCTTCAGCAACGTGACCTGGTTCCACGCCATCTCCGGCACCAATGGCACCAAGAGATTCGACAACCCCGTGCTGCCCTTCAACGACGGGGTGTACTTTGCCAGCACCGAGAAGTCCAACATCATCAGAGGCTGGATCTTCGGCACCACACTGGACAGCAAGACCCAGAGCCTGCTGATCGTGAACAACGCCACCAACGTGGTCATCAAAGTGTGCGAGTTCCAGTTCTGCAACGACCCCTTCCTGGACGTCTACTACCACAAGAACAACAAGAGCTGGATGGAAAGCGAGTTCCGGGTGTACAGCAGCGCCAACAACTGCACCTTCGAGTACGTGTCCCAGCCTTTCCTGATGGACCTGGAAGGCAAGCAGGGCAACTTCAAGAACCTGCGCGAGTTCGTGTTTAAGAACATCGACGGCTACTTCAAGATCTACAGCAAGCACACCCCTATCAACCTCGGCCGGGATCTGCCTCAGGGCTTCTCTGCTCTGGAACCCCTGGTGGATCTGCCCATCGGCATCAACATCACCCGGTTTCAGACACTGCTGGCCCTGCACAGAAGCTACCTGACACCTGGCGATAGCAGCAGCGGATGGACAGCTGGTGCCGCCGCTTACTATGTGGGCTACCTGCAGCCTAGAACCTTCCTGCTGAAGTACAACGAGAACGGCACCATCACCGACGCCGTGGATTGTGCTCTGGATCCTCTGAGCGAGACAAAGTGCACCCTGAAGTCCTTCACCGTGGAAAAGGGCATCTACCAGACCAGCAACTTCCGGGTGCAGCCCACCGAATCCATCGTGCGGTTCCCCAATATCACCAATCTGTGCCCCTTCGACGAGGTGTTCAATGCCACCACCTTCGCCTCTGTGTACGCCTGGAACCGGAAGCGGATCAGCAATTGCGTGGCCGACTACTCCGTGCTGTACAACTTCGCCCCCTTCTTCGCATTCAAGTGCTACGGCGTGTCCCCTACCAAGCTGAACGACCTGTGCTTCACAAACGTGTACGCCGACAGCTTCGTGATCCGGGGAAACGAAGTGTCACAGATTGCCCCTGGACAGACAGGCAACATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGTGTGATTGCCTGGAACAGCAACAAGCTGGACTCCACCGTCGGCGGCAACTACAATTACAGGTACCGGCTGTTCCGGAAGTCCAAGCTGAAGCCCTTCGAGCGGGACATCTCCACCGAGATCTATCAGGCCGGCAACAAGCCTTGTAACGGCGTGGCAGGCGTGAACTGCTACTTCCCACTGCAGTCCTACGGCTTTAGGCCCACATACGGCGTGGGCCACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAACTGCTGCATGCCCCTGCCACAGTGTGCGGCCCTAAGAAAAGCACCAATCTCGTGAAGAACAAATGCGTGAACTTCAACTTCAACGGCCTGACCGGCACCGGCGTGCTGACAGAGAGCAACAAGAAGTTCCTGCCATTCCAGCAGTTTGGCCGGGATATCGCCGATACCACAGACGCCGTTAGAGATCCCCAGACACTGGAAATCCTGGACATCACCCCTTGCAGCTTCGGCGGAGTGTCTGTGATCACCCCTGGCACCAACACCAGCAATCAGGTGGCAGTGCTGTACCAGGGCGTGAACTGTACCGAAGTGCCCGTGGCCATTCACGCCGATCAGCTGACACCTACATGGGGGGTGTACTCCACCGGCAGCAATGTGTTTCAGACCAGAGCCGGCTGTCTGATCGGAGCCGAGTACGTGAACAATAGCTACGAGTGCGACATCCCCATCGGCGCTGGAATCTGCGCCAGCTACCAGACACAGACAAAGAGCCACCGGAGAGCCAGAAGCGTGGCCAGCCAGAGCATCATTGCCTACACAATGTCTCTGGGCGCCGAGAACAGCGTGGCCTACTCCAACAACTCTATCGCTATCCCCACCAACTTCACCATCAGCGTGACCACAGAGATCCTGCCTGTGTCCATGACCAAGACCAGCGTGGACTGCACCATGTACATCTGCGGCGATTCCACCGAGTGCTCCAACCTGCTGCTGCAGTACGGCAGCTTCTGCACCCAGCTGAAAAGAGCCCTGACAGGGATCGCCGTGGAACAGGACAAGAACACCCAAGAGGTGTTCGCCCAAGTGAAGCAGATCTACAAGACCCCTCCTATCAAGTACTTCGGCGGCTTCAATTTCAGCCAGATTCTGCCCGATCCTAGCAAGCCCAGCAAGCGGAGCTTCATCGAGGACCTGCTGTTCAACAAAGTGACACTGGCCGACGCCGGCTTCATCAAGCAGTATGGCGATTGTCTGGGCGACATTGCCGCCAGGGATCTGATTTGCGCCCAGAAGTTTAACGGACTGACAGTGCTGCCTCCTCTGCTGACCGATGAGATGATCGCCCAGTACACATCTGCCCTGCTGGCCGGCACAATCACAAGCGGCTGGACATTTGGAGCAGGCGCCGCTCTGCAGATCCCCTTTGCTATGCAGATGGCCTACCGGTTCAACGGCATCGGAGTGACCCAGAATGTGCTGTACGAGAACCAGAAGCTGATCGCCAACCAGTTCAACAGCGCCATCGGCAAGATCCAGGACAGCCTGAGCAGCACAGCAAGCGCCCTGGGAAAGCTGCAGGACGTGGTCAACCACAATGCCCAGGCACTGAACACCCTGGTCAAGCAGCTGTCCTCCAAGTTCGGCGCCATCAGCTCTGTGCTGAACGATATCCTGAGCAGACTGGACCCTCCTGAGGCCGAGGTGCAGATCGACAGACTGATCACAGGCAGACTGCAGAGCCTCCAGACATACGTGACCCAGCAGCTGATCAGAGCCGCCGAGATTAGAGCCTCTGCCAATCTGGCCGCCACCAAGATGTCTGAGTGTGTGCTGGGCCAGAGCAAGAGAGTGGACTTTTGCGGCAAGGGCTACCACCTGATGAGCTTCCCTCAGTCTGCCCCTCACGGCGTGGTGTTTCTGCACGTGACATATGTGCCCGCTCAAGAGAAGAATTTCACCACCGCTCCAGCCATCTGCCACGACGGCAAAGCCCACTTTCCTAGAGAAGGCGTGTTCGTGTCCAACGGCACCCATTGGTTCGTGACACAGCGGAACTTCTACGAGCCCCAGATCATCACCACCGACAACACCTTCGTGTCTGGCAACTGCGACGTCGTGATCGGCATTGTGAACAATACCGTGTACGACCCTCTGCAGCCCGAGCTGGACAGCTTCAAAGAGGAACTGGACAAGTACTTTAAGAACCACACAAGCCCCGACGTGGACCTGGGCGATATCAGCGGAATCAATGCCAGCGTCGTGAACATCCAGAAAGAGATCGACCGGCTGAACGAGGTGGCCAAGAATCTGAACGAGAGCCTGATCGACCTGCAAGAACTGGGGAAGTACGAGCAGTACATCAAGTGGCCCTGGTACATCTGGCTGGGCTTTATCGCCGGACTGATTGCCATCGTGATGGTCACAATCATGCTGTGTTGCATGACCAGCTGCTGTAGCTGCCTGAAGGGCTGTTGTAGCTGTGGCAGCTGCTGCAAGTTCGACGAGGACGATTCTGAGCCCGTGCTGAAGGGCGTGAAACTGCACTACACATGATGA 103Full length RNA constructAgaauaaacuaguauucuucugguccccacagacucagagagaacccgccaccAUGUUCGUGUUCCencoding a SARS-COV-2 SUGGUGCUGCUGCCUCUGGUGUCCAGCCAGUGUGUGAACCUGAUCACCAGAACACAGUCAUACACCAprotein from an OmicronACAGCUUUACCAGAGGCGUGUACUACCCCGACAAGGUGUUCAGAUCCAGCGUGCUGCACUCUACCCBQ.1.1 variantAGGACCUGUUCCUGCCUUUCUUCAGCAACGUGACCUGGUUCCACGCCAUCUCCGGCACCAAUGGCACCAAGAGAUUCGACAACCCCGUGCUGCCCUUCAACGACGGGGUGUACUUUGCCAGCACCGAGAAGUCCAACAUCAUCAGAGGCUGGAUCUUCGGCACCACACUGGACAGCAAGACCCAGAGCCUGCUGAUCGUGAACAACGCCACCAACGUGGUCAUCAAAGUGUGCGAGUUCCAGUUCUGCAACGACCCCUUCCUGGACGUCUACUACCACAAGAACAACAAGAGCUGGAUGGAAAGCGAGUUCCGGGUGUACAGCAGCGCCAACAACUGCACCUUCGAGUACGUGUCCCAGCCUUUCCUGAUGGACCUGGAAGGCAAGCAGGGCAACUUCAAGAACCUGCGCGAGUUCGUGUUUAAGAACAUCGACGGCUACUUCAAGAUCUACAGCAAGCACACCCCUAUCAACCUCGGCCGGGAUCUGCCUCAGGGCUUCUCUGCUCUGGAACCCCUGGUGGAUCUGCCCAUCGGCAUCAACAUCACCCGGUUUCAGACACUGCUGGCCCUGCACAGAAGCUACCUGACACCUGGCGAUAGCAGCAGCGGAUGGACAGCUGGUGCCGCCGCUUACUAUGUGGGCUACCUGCAGCCUAGAACCUUCCUGCUGAAGUACAACGAGAACGGCACCAUCACCGACGCCGUGGAUUGUGCUCUGGAUCCUCUGAGCGAGACAAAGUGCACCCUGAAGUCCCUCACCGUGGAAAAGGGCAUCUACCAGACCAGCAACUUCCGGGUGCAGCCCACCGAAUCCAUCGUGCGGUUCCCCAAUAUCACCAAUCUGUGCCCCUUCGACGAGGUGUUCAAUGCCACCACCUUCGCCUCUGUGUACGCCUGGAACCGGAAGCGGAUCAGCAAUUGCGUGGCCGACUACUCCGUGCUGUACAACUUCGCCCCCUUCUUCGCAUUCAAGUGCUACGGCGUGUCCCCUACCAAGCUGAACGACCUGUGCUUCACAAACGUGUACGCCGACAGCUUCGUGAUCCGGGGAAACGAAGUGUCACAGAUUGCCCCUGGACAGACAGGCAACAUCGCCGACUACAACUACAAGCUGCCCGACGACUUCACCGGCUGUGUGAUUGCCUGGAACAGCAACAAGCUGGACUCCACCGUCGGCGGCAACUACAAUUACAGGUACCGGCUGUUCCGGAAGUCCAAGCUGAAGCCCUUCGAGCGGGACAUCUCCACCGAGAUCUAUCAGGCCGGCAACAAGCCUUGUAACGGCGUGGCAGGCGUGAACUGCUACUUCCCACUGCAGUCCUACGGCUUUAGGCCCACAUACGGCGUGGGCCACCAGCCCUACAGAGUGGUGGUGCUGAGCUUCGAACUGCUGCAUGCCCCUGCCACAGUGUGCGGCCCUAAGAAAAGCACCAAUCUCGUGAAGAACAAAUGCGUGAACUUCAACUUCAACGGCCUGACCGGCACCGGCGUGCUGACAGAGAGCAACAAGAAGUUCCUGCCAUUCCAGCAGUUUGGCCGGGAUAUCGCCGAUACCACAGACGCCGUUAGAGAUCCCCAGACACUGGAAAUCCUGGACAUCACCCCUUGCAGCUUCGGCGGAGUGUCUGUGAUCACCCCUGGCACCAACACCAGCAAUCAGGUGGCAGUGCUGUACCAGGGCGUGAACUGUACCGAAGUGCCCGUGGCCAUUCACGCCGAUCAGCUGACACCUACAUGGCGGGUGUACUCCACCGGCAGCAAUGUGUUUCAGACCAGAGCCGGCUGUCUGAUCGGAGCCGAGUACGUGAACAAUAGCUACGAGUGCGACAUCCCCAUCGGCGCUGGAAUCUGCGCCAGCUACCAGACACAGACAAAGAGCCACCGGAGAGCCAGAAGCGUGGCCAGCCAGAGCAUCAUUGCCUACACAAUGUCUCUGGGCGCCGAGAACAGCGUGGCCUACUCCAACAACUCUAUCGCUAUCCCCACCAACUUCACCAUCAGCGUGACCACAGAGAUCCUGCCUGUGUCCAUGACCAAGACCAGCGUGGACUGCACCAUGUACAUCUGCGGCGAUUCCACCGAGUGCUCCAACCUGCUGCUGCAGUACGGCAGCUUCUGCACCCAGCUGAAAAGAGCCCUGACAGGGAUCGCCGUGGAACAGGACAAGAACACCCAAGAGGUGUUCGCCCAAGUGAAGCAGAUCUACAAGACCCCUCCUAUCAAGUACUUCGGCGGCUUCAAUUUCAGCCAGAUUCUGCCCGAUCCUAGCAAGCCCAGCAAGCGGAGCUUCAUCGAGGACCUGCUGUUCAACAAAGUGACACUGGCCGACGCCGGCUUCAUCAAGCAGUAUGGCGAUUGUCUGGGCGACAUUGCCGCCAGGGAUCUGAUUUGCGCCCAGAAGUUUAACGGACUGACAGUGCUCCCUCCUCUGCUGACCGAUGAGAUGAUCGCCCAGUACACAUCUGCCCUGCUGGCCGGCACAAUCACAAGCGGCUGGACAUUUGGAGCAGGCGCCGCUCUGCAGAUCCCCUUUGCUAUGCAGAUGGCCUACCGGUUCAACGGCAUCGGAGUGACCCAGAAUGUGCUGUACGAGAACCAGAAGCUGAUCGCCAACCAGUUCAACAGCGCCAUCGGCAAGAUCCAGGACAGCCUGAGCAGCACAGCAAGCGCCCUGGGAAAGCUGCAGGACGUGGUCAACCACAAUGCCCAGGCACUGAACACCCUGGUCAAGCAGCUGUCCUCCAAGUUCGGCGCCAUCAGCUCUGUGCUGAACGAUAUCCUGAGCAGACUGGACCCUCCUGAGGCCGAGGUGCAGAUCGACAGACUGAUCACAGGCAGACUGCAGAGCCUCCAGACAUACGUGACCCAGCAGCUGAUCAGAGCCGCCGAGAUUAGAGCCUCUGCCAAUCUGGCCGCCACCAAGAUGUCUGAGUGUGUGCUGGGCCAGAGCAAGAGAGUGGACUUUUGCGGCAAGGGCUACCACCUGAUGAGCUUCCCUCAGUCUGCCCCUCACGGCGUGGUGUUUCUGCACGUGACAUAUGUGCCCGCUCAAGAGAAGAAUUUCACCACCGCUCCAGCCAUCUGCCACGACGGCAAAGCCCACUUUCCUAGAGAAGGCGUGUUCGUGUCCAACGGCACCCAUUGGUUCGUGACACAGCGGAACUUCUACGAGCCCCAGAUCAUCACCACCGACAACACCUUCGUGUCUGGCAACUGCGACGUCGUGAUCGGCAUUGUGAACAAUACCGUGUACGACCCUCUGCAGCCCGAGCUGGACAGCUUCAAAGAGGAACUGGACAAGUACUUUAAGAACCACACAAGCCCCGACGUGGACCUGGGCGAUAUCAGCGGAAUCAAUGCCAGCGUCGUGAACAUCCAGAAAGAGAUCGACCGGCUGAACGAGGUGGCCAAGAAUCUGAACGAGAGCCUGAUCGACCUGCAAGAACUGGGGAAGUACGAGCAGUACAUCAAGUGGCCCUGGUACAUCUGGCUGGGCUUUAUCGCCGGACUGAUUGCCAUCGUGAUGGUCACAAUCAUGCUGUGUUGCAUGACCAGCUGCUGUAGCUGCCUGAAGGGCUGUUGUAGCUGUGGCAGCUGCUGCAAGUUCGACGAGGACGAUUCUGAGCCCGUGCUGAAGGGCGUGAAACUGCACUACACAUGAUGAcucgagcugguacugcaugcacgcaaugcuagcugccccuuucccguccuggguaccccgagucucccccgaccucgggucccagguaugcucccaccuccaccugccccacucaccaccucugcuaguuccagacaccucccaagcacgcagcaaugcagcucaaaacgcuuagccuagccacacccccacgggaaacagcagugauuaaccuuuagcaauaaacgaaaguuuaacuaagcuauacuaaccccaggguuggucaauuucgugccagecacacccuggagcuagcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagcauaugacuaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 104Full length DNA constructAgaataaactagtattottctggtccccacagactcagagagaacccqccaccATGTTCGTGTTCCencoding a SARS-COV-2 STGGTGCTGCTGCCTCTGGTGTCCAGCCAGTGTGTGAACCTGATCACCAGAACACAGTCATACACCAprotein from an OmicronACAGCTTTACCAGAGGCGTGTACTACCCCGACAAGGTGTTCAGATCCAGCGTGCTGCACTCTACCCBQ.1.1 variantAGGACCTGTTCCTGCCTTTCTTCAGCAACGTGACCTGGTTCCACGCCATCTCCGGCACCAATGGCACCAAGAGATTCGACAACCCCGTGCTGCCCTTCAACGACGGGGTGTACTTTGCCAGCACCGAGAAGTCCAACATCATCAGAGGCTGGATCTTCGGCACCACACTGGACAGCAAGACCCAGAGCCTGCTGATCGTGAACAACGCCACCAACGTGGTCATCAAAGTGTGCGAGTTCCAGTTCTGCAACGACCCCTTCCTGGACGTCTACTACCACAAGAACAACAAGAGCTGGATGGAAAGCGAGTTCCGGGTGTACAGCAGCGCCAACAACTGCACCTTCGAGTACGTGTCCCAGCCTTTCCTGATGGACCTGGAAGGCAAGCAGGGCAACTTCAAGAACCTGCGCGAGTTCGTGTTTAAGAACATCGACGGCTACTTCAAGATCTACAGCAAGCACACCCCTATCAACCTCGGCCGGGATCTGCCTCAGGGCTTCTCTGCTCTGGAACCCCTGGTGGATCTGCCCATCGGCATCAACATCACCCGGTTTCAGACACTGCTGGCCCTGCACAGAAGCTACCTGACACCTGGCGATAGCAGCAGCGGATGGACAGCTGGTGCCGCCGCTTACTATGTGGGCTACCTGCAGCCTAGAACCTTCCTGCTGAAGTACAACGAGAACGGCACCATCACCGACGCCGTGGATTGTGCTCTGGATCCTCTGAGCGAGACAAAGTGCACCCTGAAGTCCTTCACCGTGGAAAAGGGCATCTACCAGACCAGCAACTTCCGGGTGCAGCCCACCGAATCCATCGTGCGGTTCCCCAATATCACCAATCTGTGCCCCTTCGACGAGGTGTTCAATGCCACCACCTTCGCCTCTGTGTACGCCTGGAACCGGAAGCGGATCAGCAATTGCGTGGCCGACTACTCCGTGCTGTACAACTTCGCCCCCTTCTTCGCATTCAAGTGCTACGGCGTGTCCCCTACCAAGCTGAACGACCTGTGCTTCACAAACGTGTACGCCGACAGCTTCGTGATCCGGGGAAACGAAGTGTCACAGATTGCCCCTGGACAGACAGGCAACATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGTGTGATTGCCTGGAACAGCAACAAGCTGGACTCCACCGTCGGCGGCAACTACAATTACAGGTACCGGCTGTTCCGGAAGTCCAAGCTGAAGCCCTTCGAGCGGGACATCTCCACCGAGATCTATCAGGCCGGCAACAAGCCTTGTAACGGCGTGGCAGGCGTGAACTGCTACTTCCCACTGCAGTCCTACGGCTTTAGGCCCACATACGGCGTGGGCCACCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAACTGCTGCATGCCCCTGCCACAGTGTGCGGCCCTAAGAAAAGCACCAATCTCGTGAAGAACAAATGCGTGAACTTCAACTTCAACGGCCTGACCGGCACCGGCGTGCTGACAGAGAGCAACAAGAAGTTCCTGCCATTCCAGCAGTTTGGCCGGGATATCGCCGATACCACAGACGCCGTTAGAGATCCCCAGACACTGGAAATCCTGGACATCACCCCTTGCAGCTTCGGCGGAGTGTCTGTGATCACCCCTGGCACCAACACCAGCAATCAGGTGGCAGTGCTGTACCAGGGCGTGAACTGTACCGAAGTGCCCGTGGCCATTCACGCCGATCAGCTGACACCTACATGGCGGGTGTACTCCACCGGCAGCAATGTGTTTCAGACCAGAGCCGGCTGTCTGATCGGAGCCGAGTACGTGAACAATAGCTACGAGTGCGACATCCCCATCGGCGCTGGAATCTGCGCCAGCTACCAGACACAGACAAAGAGCCACCGGAGAGCCAGAAGCGTGGCCAGCCAGAGCATCATTGCCTACACAATGTCTCTGGGCGCCGAGAACAGCGTGGCCTACTCCAACAACTCTATCGCTATCCCCACCAACTTCACCATCAGCGTGACCACAGAGATCCTGCCTGTGTCCATGACCAAGACCAGCGTGGACTGCACCATGTACATCTGCGGCGATTCCACCGAGTGCTCCAACCTGCTGCTGCAGTACGGCAGCTTCTGCACCCAGCTGAAAAGAGCCCTGACAGGGATCGCCGTGGAACAGGACAAGAACACCCAAGAGGTGTTCGCCCAAGTGAAGCAGATCTACAAGACCCCTCCTATCAAGTACTTCGGCGGCTTCAATTTCAGCCAGATTCTGCCCGATCCTAGCAAGCCCAGCAAGCGGAGCTTCATCGAGGACCTGCTGTTCAACAAAGTGACACTGGCCGACGCCGGCTTCATCAAGCAGTATGGCGATTGTCTGGGCGACATTGCCGCCAGGGATCTGATTTGCGCCCAGAAGTTTAACGGACTGACAGTGCTGCCTCCTCTGCTGACCGATGAGATGATCGCCCAGTACACATCTGCCCTGCTGGCCGGCACAATCACAAGCGGCTGGACATTTGGAGCAGGCGCCGCTCTGCAGATCCCCTTTGCTATGCAGATGGCCTACCGGTTCAACGGCATCGGAGTGACCCAGAATGTGCTGTACGAGAACCAGAAGCTGATCGCCAACCAGTTCAACAGCGCCATCGGCAAGATCCAGGACAGCCTGAGCAGCACAGCAAGCGCCCTGGGAAAGCTGCAGGACGTGGTCAACCACAATGCCCAGGCACTGAACACCCTGGTCAAGCAGCTGTCCTCCAAGTTCGGCGCCATCAGCTCTGTGCTGAACGATATCCTGAGCAGACTGGACCCTCCTGAGGCCGAGGTGCAGATCGACAGACTGATCACAGGCAGACTGCAGAGCCTCCAGACATACGTGACCCAGCAGCTGATCAGAGCCGCCGAGATTAGAGCCTCTGCCAATCTGGCCGCCACCAAGATGTCTGAGTGTGTGCTGGGCCAGAGCAAGAGAGTGGACTTTTGCGGCAAGGGCTACCACCTGATGAGCTTCCCTCAGTCTGCCCCTCACGGCGTGGTGTTTCTGCACGTGACATATGTGCCCGCTCAAGAGAAGAATTTCACCACCGCTCCAGCCATCTGCCACGACGGCAAAGCCCACTTTCCTAGAGAAGGCGTGTTCGTGTCCAACGGCACCCATTGGTTCGTGACACAGCGGAACTTCTACGAGCCCCAGATCATCACCACCGACAACACCTTCGTGTCTGGCAACTGCGACGTCGTGATCGGCATTGTGAACAATACCGTGTACGACCCTCTGCAGCCCGAGCTGGACAGCTTCAAAGAGGAACTGGACAAGTACTTTAAGAACCACACAAGCCCCGACGTGGACCTGGGCGATATCAGCGGAATCAATGCCAGCGTCGTGAACATCCAGAAAGAGATCGACCGGCTGAACGAGGTGGCCAAGAATCTGAACGAGAGCCTGATCGACCTGCAAGAACTGGGGAAGTACGAGCAGTACATCAAGTGGCCCTGGTACATCTGGCTGGGCTTTATCGCCGGACTGATTGCCATCGTGATGGTCACAATCATGCTGTGTTGCATGACCAGCTGCTGTAGCTGCCTGAAGGGCTGTTGTAGCTGTGGCAGCTGCTGCAAGTTCGACGAGGACGATTCTGAGCCCGTGCTGAAGGGCGTGAAACTGCACTACACATGATGActcgagctggtactgcatgcacgcaatgctagctgcccctttcccgtcctgggtaccccgagtctcccccgacctcgggtcccaggtatgctcccacctccacctgccccactcaccacctctgctagttccagacacctcccaagcacgcagcaatgcagctcaaaacgcttagcctagccacacccccacgggaaacagcagtgattaacctttagcaataaacgaaagtttaactaagctatactaaccccagggttggtcaatttcgtgccagccacaccctggagctagcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagcatatgactaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa

Tables 3-16 show amino acid sequences of SARS-CoV-2 S proteins encodedby RNAs described herein from different variants with 2 prolinesubstitutions at positions corresponding to K986P and V987P of SEQ IDNO: 1. In some embodiments, an RNA described herein encodes a SARS-CoV-2S protein as described herein, for example, in some embodiments, asdescribed in Tables 3-16, without the 2 proline substitutions atpositions corresponding to K986P and V987P of SEQ ID NO: 1. In someembodiments, such an RNA described herein encodes a SARS-CoV-2 S proteincomprising one or more mutations characteristic of a variant describedherein, and further comprising at least four (including, e.g., at leastfive, at least six, or more) proline mutations. In some embodiments, atleast four of such proline mutations include mutations at positionscorresponding to residues 817, 892, 899, and 942 of SEQ ID NO: 1, e.g.,as described in WO 2021243122 A2, the entire contents of which areincorporated herein by reference in its entirety. In some embodiments,such a SARS-CoV-2 S protein comprising proline substitutions atpositions corresponding to residues 817, 892, 899, and 942 of SEQ ID NO:1, may further comprise proline substitutions at positions correspondingto residues 986 and 987 of SEQ ID NO: 1.

Self-Amplifying RNA (saRNA)

The active principle of a self-amplifying RNA (saRNA) drug substance isa single-stranded RNA, which self-amplifies upon entering a cell, and acoronavirus vaccine antigen is translated thereafter. In contrast touRNA and modRNA that preferably code for a single protein, the codingregion of saRNA contains two open reading frames (ORFs). The 5′-ORFencodes an RNA-dependent RNA polymerase such as Venezuelan equineencephalitis virus (VEEV) RNA-dependent RNA polymerase (replicase). Thereplicase ORF is followed 3′ by a subgenomic promoter and a second ORFencoding an antigen. Furthermore, saRNA UTRs contain 5′ and 3′ conservedsequence elements (CSEs) required for self-amplification. The saRNAcontains common structural elements optimized for maximal efficacy ofthe RNA as the uRNA (including, e.g., 5′-cap, 5′-UTR, 3′-UTR,poly(A)-tail). In some embodiments, the saRNA preferably containsuridine. In some embodiments, the saRNA comprises one or more nucleosidemodifications as described herein. The preferred 5′ cap structure isbeta-S-ARCA(D1) (m₂ ^(7,2′-O)GppSpG).

Cytoplasmic delivery of saRNA initiates an alphavirus-like life cycle.However, saRNA does not encode for alphaviral structural proteinsrequired for genome packaging or cell entry, therefore generation ofreplication competent viral particles is very unlikely or not possible.Replication does not involve any intermediate steps that generate DNA.The use/uptake of saRNA therefore poses no risk of genomic integrationor other permanent genetic modification within the target cell.Furthermore, the saRNA itself prevents its persistent replication byeffectively activating innate immune response via recognition of dsRNAintermediates.

Different embodiments of this platform are as follows:

RBS004.1 (SEQ ID NO: 24; SEQ ID NO: 7)Structure          beta-S-ARCA(D1)-replicase-S1S2-PP-FI-A30L70 Encoded antigen    Viral spike protein (S protein) of the SARS-Cov-2(S1S2 full-length protein, sequence variant)RBS004.2 (SEQ ID NO: 25; SEQ ID NO: 7)Structure          beta-S-ARCA(D1)-replicase-S1S2-PP-FI-A30L70Encoded antigen    Viral spike protein (S protein) of the SARS-COV-2(S1S2 full-length protein, sequence variant)BNT162c1; RBS004.3 (SEQ ID NO: 26; SEQ ID NO: 5)Structure          beta-S-ARCA(D1)-replicase-RBD-GS-Fibritin-FI-A30L70Encoded antigen    Viral spike protein (S protein) of the SARS-Cov-2(partial sequence, Receptor Binding Domain (RBD) of S1S2 protein)RBS004.4 (SEQ ID NO: 27; SEQ ID NO: 28)Structure          beta-S-ARCA(D1)-replicase-RBD-GS-Fibritin-TM-FI-A30L70Encoded antigen    Viral spike protein (S protein) of the SARS-Cov-2(partial sequence, Receptor Binding Domain (RBD) of S1S2 protein)

FIG. 5 schematizes the general structure of the antigen-encoding RNAs.

In some embodiments, vaccine RNA described herein comprises a nucleotidesequence selected from the group consisting of SEQ ID NO: 15, 16, 17,19, 20, 21, 24, 25, 26, 27, 30, and 32. A particularly preferred vaccineRNA described herein comprises a nucleotide sequence selected from thegroup consisting of SEQ ID NO: 15, 17, 19, 21, 25, 26, 30, and 32 suchas selected from the group consisting of SEQ ID NO: 17, 19, 21, 26, 30,and 32.

In some embodiments, RNA described herein is formulated in lipidnanoparticles, lipoplex, polyplexes (PLX), lipidated polyplexes (LPLX),liposomes, or polysaccharide nanoparticles. In some embodiments, RNAdescribed herein is preferably formulated in lipid nanoparticles (LNP).In one embodiment, LNP comprise a cationic lipid, a neutral lipid, asteroid, a polymer conjugated lipid; and RNA. In one embodiment, thecationic lipid is ALC-0315, the neutral lipid is DSPC, the steroid ischolesterol, and the polymer conjugated lipid is ALC-0159. The preferredmode of administration is intramuscular administration, more preferablyin aqueous cryoprotectant buffer for intramuscular administration. Drugproduct is a preferably a preservative-free, sterile dispersion of RNAformulated in lipid nanoparticles (LNP) in aqueous cryoprotectant bufferfor intramuscular administration.

In different embodiments, drug product comprises the components shownbelow, preferably at the proportions or concentrations shown below:

Component Function Proportion (mol %) ALC-0315 _([1]) Functional lipid47.5 ALC-0159 _([2]) Functional lipid 1.8 DSPC _([3]) Structural lipid10.0 Cholesterol, synthetic Structural lipid 40.7 Component FunctionConcentration (mg/mL) Drug Substance Active 0.5 ALC-0315 _([1])Functional lipid 7.17 ALC-0159 _([2]) Functional lipid 0.89 DSPC _([3])Structural lipid 1.56 Cholesterol, synthetic Structural lipid 3.1Sucrose Cryoprotectant 102.69 NaCl Buffer 6.0 KCl Buffer 0.15 Na₂HPO₄Buffer 1.08 KH₂PO₄ Buffer 0.18 Water for injection Solvent/Vehicle q.s.Drug Substance Active 1.0 ALC-0315 _([1]) Functional lipid 13.56ALC-0159 _([2]) Functional lipid 1.77 DSPC _([3]) Structural lipid 3.11Cholesterol, synthetic Structural lipid 6.20 Sucrose Cryoprotectant102.69 NaCl Buffer 6.0 KCl Buffer 0.15 Na₂HPO₄ Buffer 1.08 KH₂PO₄ Buffer0.15 Water for injection Solvent/Vehicle q.s. _([1]) ALC-0315 =((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)/6-[N-6-(2-hexyldecanoyloxy)hexyl-N-(4-hydroxybutyl)amino]hexyl2-hexyldecanoate _([2]) ALC-0159 = 2-[(polyethyleneglycol)-2000]-N,N-ditetradecylacetamide/2-[2-(ω-methoxy(polyethyleneglycol2000)ethoxy]-N,N-ditetradecylacetamide_([3]) DSPC = 1,2-Distearoyl-sn-glycero-3-phosphocholine q.s. = quantumsatis (as much as may suffice)

In some embodiments, particles disclosed herein are formulated in asolution comprising 10 mM Tris and 10% sucrose, and optionally having apH of about 7.4. In some embodiments, particles disclosed herein areformulated in a solution comprising about 103 mg/ml sucrose, about 0.20mg/ml tromethamine (Tris base), and about 1.32 mg/ml Tris.

In some embodiments, a composition comprises:

-   -   (a) about 0.1 mg/mL RNA comprising an open reading frame        encoding a polypeptide that comprises a SARS-CoV-2 protein or an        immunogenic fragment or variant thereof,    -   (b) about 1.43 mg/ml ALC-0315,    -   (c) about 0.18 mg/ml ALC-0159,    -   (d) about 0.31 mg/ml DSPC,    -   (e) about 0.62 mg/ml cholesterol,    -   (f) about 103 mg/ml sucrose,    -   (g) about 0.20 mg/ml tromethamine (Tris base),    -   (h) about 1.32 mg/ml Tris (hydroxymethyl) aminomethane        hydrochloride (Tris HCl), and    -   (i) q.s. water.

In one embodiment, the ratio of RNA (e.g., mRNA) to total lipid (N/P) isbetween 6.0 and 6.5 such as about 6.0 or about 6.3.

Nucleic Acid Containing Particles

Nucleic acids described herein such as RNA encoding a vaccine antigenmay be administered formulated as particles.

In the context of the present disclosure, the term “particle” relates toa structured entity formed by molecules or molecule complexes. In oneembodiment, the term “particle” relates to a micro- or nano-sizedstructure, such as a micro- or nano-sized compact structure dispersed ina medium. In one embodiment, a particle is a nucleic acid containingparticle such as a particle comprising DNA, RNA or a mixture thereof.

Electrostatic interactions between positively charged molecules such aspolymers and lipids and negatively charged nucleic acid are involved inparticle formation. This results in complexation and spontaneousformation of nucleic acid particles. In one embodiment, a nucleic acidparticle is a nanoparticle.

As used in the present disclosure, “nanoparticle” refers to a particlehaving an average diameter suitable for parenteral administration.

A “nucleic acid particle” can be used to deliver nucleic acid to atarget site of interest (e.g., cell, tissue, organ, and the like). Anucleic acid particle may be formed from at least one cationic orcationically ionizable lipid or lipid-like material, at least onecationic polymer such as protamine, or a mixture thereof and nucleicacid. In some embodiments, exemplary nanoparticles include lipidnanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), liposomes,or polysaccharide nanoparticles.

In some embodiments, the RNA encoding an amino acid sequence comprisinga SARS-CoV-2 S protein, an immunogenic variant thereof, or animmunogenic fragment of the SARS-CoV-2 S protein or the immunogenicvariant thereof is formulated as LNPs. In some embodiments, the LNPscomprise one or more cationically ionizable lipids; one or more neutrallipids (e.g., in some embodiments sterol such as, e.g., cholesterol;and/or phospholipids), and one or more polymer-conjugated lipids. Insome embodiments, the formulation comprises ALC-0315(4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate),ALC-0159 (2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide),DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), cholesterol, sucrose,trometamol (Tris), trometamol hydrochloride and water.

RNA particles described herein include nanoparticles. In someembodiments, exemplary nanoparticles include lipid nanoparticles,lipoplex, polyplexes (PLX), lipidated polyplexes (LPLX), liposomes, orpolysaccharide nanoparticles.

Polyplexes (PLX), polysaccharide nanoparticles, and liposomes, are alldelivery technologies that are well known to a person of skill in theart. See, e.g., Lächelt, Ulrich, and Ernst Wagner. “Nucleic acidtherapeutics using polyplexes: a journey of 50 years (and beyond)”Chemical reviews 115.19 (2015): 11043-11078; Plucinski, Alexander, ZanLyu, and Bernhard VKJ Schmidt, “Polysaccharide nanoparticles: fromfabrication to applications.” Journal of Materials Chemistry B (2021);and Tenchov, Rumiana, et al. “Lipid Nanoparticles—From Liposomes to mRNAVaccine Delivery, a Landscape of Research Diversity and Advancement,”ACS nano 15.11 (2021): 16982-17015, respectively, the contents of eachof which are hereby incorporated by reference herein in their entirety.

In some embodiments, the concentration of the RNA in the pharmaceuticalRNA preparation is about 0.1 mg/ml. In some embodiments, theconcentration of the RNA in the pharmaceutical RNA preparation is about30 μg/ml to about 100 μg/ml. In some embodiments, the concentration ofthe RNA in the pharmaceutical RNA preparation is about 50 μg/ml to about100 μg/ml.

Without intending to be bound by any theory, it is believed that thecationic or cationically ionizable lipid or lipid-like material and/orthe cationic polymer combine together with the nucleic acid to formaggregates, and this aggregation results in colloidally stableparticles.

In one embodiment, particles described herein further comprise at leastone lipid or lipid-like material other than a cationic or cationicallyionizable lipid or lipid-like material, at least one polymer other thana cationic polymer, or a mixture thereof

In some embodiments, nucleic acid particles comprise more than one typeof nucleic acid molecules, where the molecular parameters of the nucleicacid molecules may be similar or different from each other, like withrespect to molar mass or fundamental structural elements such asmolecular architecture, capping, coding regions or other features.

Nucleic acid particles described herein may have an average diameterthat in one embodiment ranges from about 30 nm to about 1000 nm, fromabout 50 nm to about 800 nm, from about 70 nm to about 600 nm, fromabout 90 nm to about 400 nm, or from about 100 nm to about 300 nm.

Nucleic acid particles described herein may exhibit a polydispersityindex less than about 0.5, less than about 0.4, less than about 0.3, orabout 0.2 or less. By way of example, the nucleic acid particles canexhibit a polydispersity index in a range of about 0.1 to about 0.3 orabout 0.2 to about 0.3.

With respect to RNA lipid particles, the N/P ratio gives the ratio ofthe nitrogen groups in the lipid to the number of phosphate groups inthe RNA. It is correlated to the charge ratio, as the nitrogen atoms(depending on the pH) are usually positively charged and the phosphategroups are negatively charged. The N/P ratio, where a charge equilibriumexists, depends on the pH. Lipid formulations are frequently formed atN/P ratios larger than four up to twelve, because positively chargednanoparticles are considered favorable for transfection. In that case,RNA is considered to be completely bound to nanoparticles.

Nucleic acid particles described herein can be prepared using a widerange of methods that may involve obtaining a colloid from at least onecationic or cationically ionizable lipid or lipid-like material and/orat least one cationic polymer and mixing the colloid with nucleic acidto obtain nucleic acid particles.

The term “colloid” as used herein relates to a type of homogeneousmixture in which dispersed particles do not settle out. The insolubleparticles in the mixture are microscopic, with particle sizes between 1and 1000 nanometers. The mixture may be termed a colloid or a colloidalsuspension. Sometimes the term “colloid” only refers to the particles inthe mixture and not the entire suspension.

For the preparation of colloids comprising at least one cationic orcationically ionizable lipid or lipid-like material and/or at least onecationic polymer methods are applicable herein that are conventionallyused for preparing liposomal vesicles and are appropriately adapted. Themost commonly used methods for preparing liposomal vesicles share thefollowing fundamental stages: (i) lipids dissolution in organicsolvents, (ii) drying of the resultant solution, and (iii) hydration ofdried lipid (using various aqueous media).

In the film hydration method, lipids are firstly dissolved in a suitableorganic solvent, and dried down to yield a thin film at the bottom ofthe flask. The obtained lipid film is hydrated using an appropriateaqueous medium to produce a liposomal dispersion. Furthermore, anadditional downsizing step may be included.

Reverse phase evaporation is an alternative method to the film hydrationfor preparing liposomal vesicles that involves formation of awater-in-oil emulsion between an aqueous phase and an organic phasecontaining lipids. A brief sonication of this mixture is required forsystem homogenization. The removal of the organic phase under reducedpressure yields a milky gel that turns subsequently into a liposomalsuspension.

The term “ethanol injection technique” refers to a process, in which anethanol solution comprising lipids is rapidly injected into an aqueoussolution through a needle. This action disperses the lipids throughoutthe solution and promotes lipid structure formation, for example lipidvesicle formation such as liposome formation. Generally, the RNAlipoplex particles described herein are obtainable by adding RNA to acolloidal liposome dispersion.

Using the ethanol injection technique, such colloidal liposomedispersion is, in one embodiment, formed as follows: an ethanol solutioncomprising lipids, such as cationic lipids and additional lipids, isinjected into an aqueous solution under stirring. In one embodiment, theRNA lipoplex particles described herein are obtainable without a step ofextrusion.

The term “extruding” or “extrusion” refers to the creation of particleshaving a fixed, cross-sectional profile. In particular, it refers to thedownsizing of a particle, whereby the particle is forced through filterswith defined pores.

Other methods having organic solvent free characteristics may also beused according to the present disclosure for preparing a colloid.

LNPs typically comprise four components: ionizable cationic lipids,neutral lipids such as phospholipids, a steroid such as cholesterol, anda polymer conjugated lipid such as polyethylene glycol (PEG)-lipids.Each component is responsible for payload protection, and enableseffective intracellular delivery. LNPs may be prepared by mixing lipidsdissolved in ethanol rapidly with nucleic acid in an aqueous buffer.

The term “average diameter” refers to the mean hydrodynamic diameter ofparticles as measured by dynamic laser light scattering (DLS) with dataanalysis using the so-called cumulant algorithm, which provides asresults the so-called Z_(average) with the dimension of a length, andthe polydispersity index (PI), which is dimensionless (Koppel, D., J.Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321). Here “average diameter”,“diameter” or “size” for particles is used synonymously with this valueof the Z_(average).

The “polydispersity index” is preferably calculated based on dynamiclight scattering measurements by the so-called cumulant analysis asmentioned in the definition of the “average diameter”. Under certainprerequisites, it can be taken as a measure of the size distribution ofan ensemble of nanoparticles.

Different types of nucleic acid containing particles have been describedpreviously to be suitable for delivery of nucleic acid in particulateform (e.g. Kaczmarek, J. C. et al., 2017, Genome Medicine 9, 60). Fornon-viral nucleic acid delivery vehicles, nanoparticle encapsulation ofnucleic acid physically protects nucleic acid from degradation and,depending on the specific chemistry, can aid in cellular uptake andendosomal escape.

The present disclosure describes particles comprising nucleic acid, atleast one cationic or cationically ionizable lipid or lipid-likematerial, and/or at least one cationic polymer which associate withnucleic acid to form nucleic acid particles and compositions comprisingsuch particles. The nucleic acid particles may comprise nucleic acidwhich is complexed in different forms by non-covalent interactions tothe particle. The particles described herein are not viral particles, inparticular infectious viral particles, i.e., they are not able tovirally infect cells. Suitable cationic or cationically ionizable lipidsor lipid-like materials and cationic polymers are those that formnucleic acid particles and are included by the term “particle formingcomponents” or “particle forming agents”. The term “particle formingcomponents” or “particle forming agents” relates to any components whichassociate with nucleic acid to form nucleic acid particles. Suchcomponents include any component which can be part of nucleic acidparticles.

In some embodiments, a nucleic acid containing particle (e.g., a lipidnanoparticle (LNP)) comprises two or more RNA molecules, each comprisinga different nucleic acid sequence. In some embodiments, a nucleic acidcontaining particle comprises two or more RNA molecules, each encoding adifferent immunogenic polypeptide or immunogenic fragment thereof. Insome embodiments, two or more RNA molecules present in a nucleic acidcontaining particle comprise: a first RNA molecule encodes animmunogenic polypeptide or immunogenic fragment thereof from acoronavirus and a second RNA molecule encodes an immunogenic polypeptideor immunogenic fragment thereof from an infectious disease pathogen(e.g., virus, bacteria, parasite, etc.). For example, in someembodiments, two or more RNA molecules present in a nucleic acidcontaining particle comprise: a first RNA molecule encoding animmunogenic polypeptide or immunogenic fragment thereof from acoronavirus (e.g., in some embodiments SARS-CoV-2 Wuhan strain or avariant thereof, e.g., a SARS-CoV-2 having one or more mutationscharacteristic of an Omicron variant) and a second RNA molecule encodingan immunogenic polypeptide or immunogenic fragment thereof from aninfluenza virus. In some embodiments, two or more RNA molecules presentin a nucleic acid containing particle comprise: a first RNA moleculeencoding an immunogenic polypeptide or immunogenic fragment thereof froma first coronavirus (e.g., as described herein) and a second RNAmolecule encoding an immunogenic polypeptide or immunogenic fragmentthereof from a second coronavirus (e.g., as described herein). In someembodiments, a first coronavirus is different from a second coronavirus.In some embodiments, a first and/or second coronavirus is independentlyfrom a SARS-CoV-2 Wuhan strain or a variant thereof, e.g., a SARS-CoV-2having one or more mutations characteristic of an Omicron variant.

In some embodiments, two or more RNA molecules present in a nucleic acidcontaining particle each encode an immunogenic polypeptide or animmunogenic fragment thereof from the same and/or different strainsand/or variants of coronavirus (e.g., in some embodiments SARS-CoV-2strains or variants). For example, in some embodiments, two or more RNAmolecules present in a nucleic acid containing particle each encode adifferent immunogenic polypeptide or immunogenic fragment thereof from acoronavirus membrane protein, a coronavirus nucleocapsid protein, acoronavirus spike protein, a coronavirus non-structural protein and/or acoronavirus accessory protein. In some embodiments, such immunogenicpolypeptides or immunogenic fragments thereof may be from the same or adifferent coronavirus (e.g., in some embodiments a SARS-CoV-2 Wuhanstrain or variants thereof, for example, in some embodiments a varianthaving one or more mutations characteristic of a prevalent variant suchas an Omicron variant). In some embodiments, a nucleic acid containingparticle comprises a first RNA molecule encoding a SARS-CoV-2 S proteinor an immunogenic fragment thereof from a first strain or variant, and asecond RNA molecule encoding a SARS-CoV-2 S protein or an immunogenicfragment thereof from a second strain or variant, wherein the secondstrain or variant is different from the first strain or variant.

In some embodiments, a nucleic acid containing particle (e.g., in someembodiments an LNP as described herein) comprises a first RNA moleculeencoding a SARS-CoV-2 S protein from a Wuhan strain and a second RNAmolecule encoding a SARS-CoV-2 S protein comprising one or moremutations that are characteristic of an Omicron variant (e.g., a BA.1,BA.2, BA.3, BA.4, or BA.5 Omicron variant).

In some embodiments, a nucleic acid containing particle comprises afirst RNA molecule encoding a SARS-CoV-2 S protein from a Wuhan strainand a second RNA molecule encoding a SARS-CoV-2 S protein comprising oneor more mutations that are characteristic of an Omicron BA.1 variant. Insome embodiments, the ratio of the first RNA molecule encoding aSARS-CoV-2 S protein from a Wuhan strain and the second RNA moleculeencoding a SARS-CoV-2 S protein comprising one or more mutations thatare characteristic of an Omicron BA.1 variant is 1:1. In someembodiments, the ratio of the first RNA molecule encoding a SARS-CoV-2 Sprotein from a Wuhan strain and the second RNA molecule encoding aSARS-CoV-2 S protein comprising one or more mutations that arecharacteristic of an Omicron BA.1 variant is 1:2. In some embodiments,the ratio of the first RNA molecule encoding a SARS-CoV-2 S protein froma Wuhan strain and the second RNA molecule encoding a SARS-CoV-2 Sprotein comprising one or more mutations that are characteristic of anOmicron BA.1 variant is 1:3. In some embodiments, a nucleic acidcontaining particle comprises a first RNA molecule encoding a SARS-CoV-2S protein from a Wuhan strain and a second RNA molecule encoding aSARS-CoV-2 S protein comprising one or more mutations that arecharacteristic of an Omicron BA.2 variant. In some embodiments, theratio of the first RNA molecule encoding a SARS-CoV-2 S protein from aWuhan strain and the second RNA molecule encoding a SARS-CoV-2 S proteincomprising one or more mutations that are characteristic of an OmicronBA.2 variant is 1:1. In some embodiments, the ratio of the first RNAmolecule encoding a SARS-CoV-2 S protein from a Wuhan strain and thesecond RNA molecule encoding a SARS-CoV-2 S protein comprising one ormore mutations that are characteristic of an Omicron BA.2 variant is1:2. In some embodiments, the ratio of the first RNA molecule encoding aSARS-CoV-2 S protein from a Wuhan strain and the second RNA moleculeencoding a SARS-CoV-2 S protein comprising one or more mutations thatare characteristic of an Omicron BA.2 variant is 1:3. In someembodiments, a nucleic acid containing particle comprises a first RNAmolecule encoding a SARS-CoV-2 S protein from a Wuhan strain and asecond RNA molecule encoding a SARS-CoV-2 S protein comprising one ormore mutations that are characteristic of an Omicron BA.3 variant. Insome embodiments, a nucleic acid containing particle comprises a firstRNA molecule encoding a SARS-CoV-2 S protein from a Wuhan strain and asecond RNA molecule encoding a SARS-CoV-2 S protein comprising one ormore mutations that are characteristic of an Omicron BA.4 or BA.5variant. In some embodiments, the ratio of the first RNA moleculeencoding a SARS-CoV-2 S protein from a Wuhan strain and the second RNAmolecule encoding a SARS-CoV-2 S protein comprising one or moremutations that are characteristic of an Omicron BA.4 or BA.5 variant is1:1. In some embodiments, the ratio of the first RNA molecule encoding aSARS-CoV-2 S protein from a Wuhan strain and the second RNA moleculeencoding a SARS-CoV-2 S protein comprising one or more mutations thatare characteristic of an Omicron BA.4 or BA.5 variant is 1:2. In someembodiments, the ratio of the first RNA molecule encoding a SARS-CoV-2 Sprotein from a Wuhan strain and the second RNA molecule encoding aSARS-CoV-2 S protein comprising one or more mutations that arecharacteristic of an Omicron BA.4 or BA.5 variant is 1:3.

In some embodiments, a nucleic acid containing particle comprises afirst RNA molecule encoding a SARS-CoV-2 S protein from a first Omicronvariant and a second RNA molecule encoding a SARS-CoV-2 S proteincomprising one or more mutations that are characteristic of a secondOmicron variant.

In some embodiments, a nucleic acid containing particle comprises afirst RNA molecule encoding a SARS-CoV-2 S protein from a BA.1 Omicronvariant and a second RNA molecule encoding a SARS-CoV-2 S proteincomprising one or more mutations that are characteristic of a BA.2Omicron variant. In some embodiments, the ratio of the first RNAmolecule encoding a SARS-CoV-2 S protein comprising one or moremutations characteristic of a BA.1 Omicron variant strain and the secondRNA molecule encoding a SARS-CoV-2 S protein comprising one or moremutations that are characteristic of an Omicron BA.2 variant is 1:1. Insome embodiments, the ratio of the first RNA molecule encoding aSARS-CoV-2 S protein comprising one or more mutations characteristic ofa BA.1 Omicron variant strain and the second RNA molecule encoding aSARS-CoV-2 S protein comprising one or more mutations that arecharacteristic of an Omicron BA.2 variant is 1:2. In some embodiments,the ratio of the first RNA molecule encoding a SARS-CoV-2 S proteincomprising one or more mutations characteristic of a BA.1 Omicronvariant strain and the second RNA molecule encoding a SARS-CoV-2 Sprotein comprising one or more mutations that are characteristic of anOmicron BA.2 variant is 1:3.

In some embodiments, a nucleic acid containing particle comprises afirst RNA molecule encoding a SARS-CoV-2 S protein from a BA.1 Omicronvariant and a second RNA molecule encoding a SARS-CoV-2 S proteincomprising one or more mutations that are characteristic of a BA.3Omicron variant. In some embodiments, the ratio of the first RNAmolecule encoding a SARS-CoV-2 S protein comprising one or moremutations characteristic of a BA.1 Omicron variant strain and the secondRNA molecule encoding a SARS-CoV-2 S protein comprising one or moremutations that are characteristic of an Omicron BA.3 variant is 1:1. Insome embodiments, the ratio of the first RNA molecule encoding aSARS-CoV-2 S protein comprising one or more mutations characteristic ofa BA.1 Omicron variant strain and the second RNA molecule encoding aSARS-CoV-2 S protein comprising one or more mutations that arecharacteristic of an Omicron BA.3 variant is 1:2. In some embodiments,the ratio of the first RNA molecule encoding a SARS-CoV-2 S proteincomprising one or more mutations characteristic of a BA.1 Omicronvariant strain and the second RNA molecule encoding a SARS-CoV-2 Sprotein comprising one or more mutations that are characteristic of anOmicron BA.3 variant is 1:3.

In some embodiments, a nucleic acid containing particle comprises afirst RNA molecule encoding a SARS-CoV-2 S protein from a BA.1 Omicronvariant and a second RNA molecule encoding a SARS-CoV-2 S proteincomprising one or more mutations that are characteristic of a BA.4 orBA.5 Omicron variant. In some embodiments, the ratio of the first RNAmolecule encoding a SARS-CoV-2 S protein comprising one or moremutations characteristic of a BA.1 Omicron variant strain and the secondRNA molecule encoding a SARS-CoV-2 S protein comprising one or moremutations that are characteristic of an Omicron BA.4 or BA.5 variant is1:1. In some embodiments, the ratio of the first RNA molecule encoding aSARS-CoV-2 S protein comprising one or more mutations characteristic ofa BA.1 Omicron variant strain and the second RNA molecule encoding aSARS-CoV-2 S protein comprising one or more mutations that arecharacteristic of an Omicron BA.4 or BA.5 variant is 1:2. In someembodiments, the ratio of the first RNA molecule encoding a SARS-CoV-2 Sprotein comprising one or more mutations characteristic of a BA.1Omicron variant strain and the second RNA molecule encoding a SARS-CoV-2S protein comprising one or more mutations that are characteristic of anOmicron BA.4 or BA.5 variant is 1:3.

In some embodiments, a nucleic acid containing particle comprises afirst RNA molecule encoding a SARS-CoV-2 S protein from a BA.2 Omicronvariant and a second RNA molecule encoding a SARS-CoV-2 S proteincomprising one or more mutations that are characteristic of a BA.3Omicron variant. In some embodiments, the ratio of the first RNAmolecule encoding a SARS-CoV-2 S protein comprising one or moremutations characteristic of a BA.2 Omicron variant strain and the secondRNA molecule encoding a SARS-CoV-2 S protein comprising one or moremutations that are characteristic of an Omicron BA.3 variant is 1:1. Insome embodiments, the ratio of the first RNA molecule encoding aSARS-CoV-2 S protein comprising one or more mutations characteristic ofa BA.2 Omicron variant strain and the second RNA molecule encoding aSARS-CoV-2 S protein comprising one or more mutations that arecharacteristic of an Omicron BA.3 variant is 1:2. In some embodiments,the ratio of the first RNA molecule encoding a SARS-CoV-2 S proteincomprising one or more mutations characteristic of a BA.2 Omicronvariant strain and the second RNA molecule encoding a SARS-CoV-2 Sprotein comprising one or more mutations that are characteristic of anOmicron BA.3 variant is 1:3.

In some embodiments, a nucleic acid containing particle comprises afirst RNA molecule encoding a SARS-CoV-2 S protein from a BA.2 Omicronvariant and a second RNA molecule encoding a SARS-CoV-2 S proteincomprising one or more mutations that are characteristic of a BA.4 orBA.5 Omicron variant. In some embodiments, the ratio of the first RNAmolecule encoding a SARS-CoV-2 S protein comprising one or moremutations characteristic of a BA.2 Omicron variant strain and the secondRNA molecule encoding a SARS-CoV-2 S protein comprising one or moremutations that are characteristic of an Omicron BA.4 or BA.5 variant is1:1. In some embodiments, the ratio of the first RNA molecule encoding aSARS-CoV-2 S protein comprising one or more mutations characteristic ofa BA.2 Omicron variant strain and the second RNA molecule encoding aSARS-CoV-2 S protein comprising one or more mutations that arecharacteristic of an Omicron BA.4 or BA.5 variant is 1:2. In someembodiments, the ratio of the first RNA molecule encoding a SARS-CoV-2 Sprotein comprising one or more mutations characteristic of a BA.2Omicron variant strain and the second RNA molecule encoding a SARS-CoV-2S protein comprising one or more mutations that are characteristic of anOmicron BA.4 or BA.5 variant is 1:3.

In some embodiments, a nucleic acid containing particle comprises afirst RNA molecule encoding a SARS-CoV-2 S protein from a BA.3 Omicronvariant and a second RNA molecule encoding a SARS-CoV-2 S proteincomprising one or more mutations that are characteristic of a BA.4 orBA.5 Omicron variant. In some embodiments, the ratio of the first RNAmolecule encoding a SARS-CoV-2 S protein comprising one or moremutations characteristic of a BA.3 Omicron variant strain and the secondRNA molecule encoding a SARS-CoV-2 S protein comprising one or moremutations that are characteristic of an Omicron BA.4 or BA.5 variant is1:1. In some embodiments, the ratio of the first RNA molecule encoding aSARS-CoV-2 S protein comprising one or more mutations characteristic ofa BA.3 Omicron variant strain and the second RNA molecule encoding aSARS-CoV-2 S protein comprising one or more mutations that arecharacteristic of an Omicron BA.4 or BA.5 variant is 1:2. In someembodiments, the ratio of the first RNA molecule encoding a SARS-CoV-2 Sprotein comprising one or more mutations characteristic of a BA.3Omicron variant strain and the second RNA molecule encoding a SARS-CoV-2S protein comprising one or more mutations that are characteristic of anOmicron BA.4 or BA.5 variant is 1:3.

In some embodiments, a nucleic acid containing particle (e.g., in someembodiments an LNP as described herein) comprises three or more RNAmolecules, each encoding a SARS-CoV-2 S protein comprising mutations ofa different SARS-CoV-2 variant. In some embodiments, a nucleic acidcontaining particle comprises a first RNA molecule encoding a SARS-CoV-2S protein from a Wuhan strain, a second RNA molecule encoding aSARS-CoV-2 S protein comprising one or more mutations that arecharacteristic of an Omicron variant (e.g., a BA.1, BA.2, BA.3, BA.4, orBA.5 Omicron variant), and a third RNA molecule encoding a SARS-CoV-2 Sprotein comprising one or more mutations that are characteristic of anOmicron variant (e.g., a BA.1, BA.2, BA.3, BA.4, or BA.5 Omicronvariant), wherein the second and third RNA molecules encode a SARS-CoV-2S protein comprising one or mutations characteristic of differentOmicron subvariants. In some embodiments, a nucleic acid containingparticle comprises a first RNA molecule encoding a SARS-CoV-2 S proteinfrom a Wuhan strain, a second RNA molecule encoding a SARS-CoV-2 Sprotein comprising one or more mutations that are characteristic of aBA.1 Omicron variant, and a third RNA molecule encoding a SARS-CoV-2 Sprotein comprising one or more mutations that are characteristic of aBA.2 Omicron variant. In some embodiments, a nucleic acid containingparticle comprises a first RNA molecule encoding a SARS-CoV-2 S proteinfrom a Wuhan strain, a second RNA molecule encoding a SARS-CoV-2 Sprotein comprising one or more mutations that are characteristic of aBA.1 Omicron variant, and a third RNA molecule encoding a SARS-CoV-2 Sprotein comprising one or more mutations that are characteristic of aBA.2 Omicron variant. In some embodiments, a nucleic acid containingparticle comprises a first RNA molecule encoding a SARS-CoV-2 S proteinfrom a Wuhan strain, a second RNA molecule encoding a SARS-CoV-2 Sprotein comprising one or more mutations that are characteristic of aBA.1 Omicron variant, and a third RNA molecule encoding a SARS-CoV-2 Sprotein comprising one or more mutations that are characteristic of aBA.4/5 Omicron variant. In some embodiments, a nucleic acid containingparticle comprises a first RNA molecule encoding a SARS-CoV-2 S proteinfrom a Wuhan strain, a second RNA molecule encoding a SARS-CoV-2 Sprotein comprising one or more mutations that are characteristic of aBA.2 Omicron variant, and a third RNA molecule encoding a SARS-CoV-2 Sprotein comprising one or more mutations that are characteristic of aBA.4/5 Omicron variant. In some embodiments, a nucleic acid containingparticle comprises a first RNA molecule encoding a SARS-CoV-2 S proteincomprising one or more mutations that are characteristic of a BA.1Omicron variant, a second RNA molecule encoding a SARS-CoV-2 S proteincomprising one or more mutations that are characteristic of a BA.2Omicron variant, and a third RNA molecule encoding a SARS-CoV-2 Sprotein comprising one or more mutations that are characteristic of aBA.4/5 Omicron variant.

In some embodiments, a nucleic acid containing particle (e.g., in someembodiments an LNP as described herein) comprising two or more RNAmolecules, comprises each RNA molecule in the same amount (i.e., at a1:1 ratio).

In some embodiments, a nucleic acid containing particle (e.g., in someembodiments an LNP as described herein) comprising two or more RNAmolecules, comprises a different amount of each RNA molecule. Forexample, in some embodiments, a nucleic acid containing particlecomprises a first RNA molecule and a second RNA molecule, where thefirst RNA molecule is present in an amount that is 0.01 to 100 timesthat of the second RNA molecule (e.g., wherein the amount of the firstRNA molecule is 0.01 to 50, 0.01 to 4, 0.01 to 30, 0.01 to 25, 0.01 to20, 0.01 to 15, 0.01 to 10, 0.01 to 9, 0.01 to 8, 0.01 to 7, 0.01 to 6,0.01 to 5, 0.01 to 4, 0.01 to 3, 0.01 to 2, 0.01 to 1.5, 1 to 50, 1 to4, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7,1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 to 1.5 times higher thanthe second RNA molecule). In some embodiments, a nucleic acid containingparticle comprises a first RNA molecule and a second RNA molecule,wherein the concentration of the first RNA molecule is 1 to 10 timesthat of the second RNA molecule. In some embodiments, a nucleic acidcontaining particle comprises a first RNA molecule and a second RNAmolecule, wherein the concentration of the first RNA molecule is 1 to 5times that of the second RNA molecule. In some embodiments, a nucleicacid containing particle comprises a first RNA molecule and a second RNAmolecule, wherein the concentration of the first RNA molecule is 1 to 3times that of the second RNA molecule. In some embodiments, a nucleicacid containing particle comprises a first RNA molecule and a second RNAmolecule, wherein the concentration of the first RNA molecule is 2 timesthat of the second RNA molecule. In some embodiments, a nucleic acidcontaining particle comprises a first RNA molecule and a second RNAmolecule, wherein the concentration of the first RNA molecule is 3 timesthat of the second RNA molecule.

In some embodiments, a nucleic acid containing particle (e.g., in someembodiments an LNP as described herein) comprising three RNA molecules,comprises each RNA molecule in the same amount (i.e., at a 1:1:1 ratio).

In some embodiments, a nucleic acid containing particle (e.g., in someembodiments an LNP as described herein) comprising three RNA molecules,comprises a different amount of each RNA molecule. For example, in someembodiments, the ratio of first RNA molecule:second RNA molecule:thirdRNA molecule is 1:0.01-100:0.01-100 (e.g., 1:0.01-50:0.01-50;1:0.01-40:0.01-40; 1:0.01-30:0.01-25; 1:0.01-25:0.01-25;1:0.01-20:0.01-20; 1:0.01-15:0.01-15; 1:0.01-10:0.01-9; 1:0.01-9:0.01-9;1:0.01-8:0.01-8; 1:0.01-7:0.01-7; 1:0.01-6:0.01-6; 1:0.01-5:0.01-5;1:0.01-4:0.01-4; 1:0.01-3:0.01-3; 1:0.01-2:0.01-2; or1:0.01-1.5:0.01-1.5).

In some embodiments, the ratio of first RNA molecule:second RNAmolecule:third RNA molecule is 1:1:3. In some embodiments, the ratio offirst RNA molecule:second RNA molecule:third RNA molecule is 1:3:3.

In some embodiments, a first RNA molecule encoding a SARS-CoV-2 Sprotein from a Wuhan strain comprises a nucleotide sequence that encodesthe amino acid sequence of SEQ ID NO: 7. In some embodiments, a firstRNA molecule encoding a SARS-CoV-2 S protein from a Wuhan straincomprises a nucleotide sequence that is at least 80% identical (e.g., atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identical) to SEQ ID NO: 9. In some embodiments, a first RNAmolecule encoding a SARS-COV-2 S protein from a Wuhan strain comprises anucleotide sequence that is at least 80% identical to (e.g., at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% identical to) SEQ ID NO: 20. In some embodiments, a first RNAmolecule encoding a SARS-COV-2 S protein from a Wuhan strain comprises anucleotide sequence that encodes an amino acid sequence that is at least80% identical to (e.g., at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% identical to) SEQ ID NO: 7. Insome embodiments, a second RNA molecule encoding a SARS-CoV-2 S proteinhaving one or more mutations that are characteristic of an Omicronvariant comprises a nucleotide sequence that encodes the amino acidsequence of SEQ ID NO: 49. In some embodiments, a second RNA moleculeencoding a SARS-CoV-2 S protein comprising one or more mutationscharacteristic of an Omicron variant comprises a nucleotide sequencethat is at least 80% identical (e.g., at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical) to SEQID NO: 50. In some embodiments, a second RNA molecule encoding aSARS-COV-2 S protein comprising one or more mutations characteristic ofan Omicron variant comprises a nucleotide sequence that is at least 80%identical to (e.g., at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identical to) SEQ ID NO: 51. In someembodiments, a second RNA molecule encoding a SARS-COV-2 S proteincomprising one or more mutations characteristic of an Omicron variantcomprises a nucleotide sequence that encodes an amino acid sequence thatis at least 80% identical to (e.g., at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identical to) SEQ IDNO: 49.

In some embodiments, a second RNA molecule encoding a SARS-CoV-2 Sprotein having one or more mutations that are characteristic of anOmicron variant comprises a nucleotide sequence that encodes the aminoacid sequence of SEQ ID NO: 64. In some embodiments, a second RNAmolecule encoding a SARS-CoV-2 S protein comprising one or moremutations characteristic of an Omicron variant comprises a nucleotidesequence that is at least 80% identical (e.g., at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99%identical) to SEQ ID NO: 65. In some embodiments, a second RNA moleculeencoding a SARS-COV-2 S protein comprising one or more mutationscharacteristic of an Omicron variant comprises a nucleotide sequencethat is at least 80% identical to (e.g., at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical to) SEQID NO: 67. In some embodiments, a second RNA molecule encoding aSARS-COV-2 S protein comprising one or more mutations characteristic ofan Omicron variant comprises a nucleotide sequence that encodes an aminoacid sequence that is at least 80% identical to (e.g., at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to) SEQ ID NO: 64.

In some embodiments, a second RNA molecule encoding a SARS-CoV-2 Sprotein having one or more mutations that are characteristic of anOmicron variant comprises a nucleotide sequence that encodes the aminoacid sequence of SEQ ID NO: 69. In some embodiments, a second RNAmolecule encoding a SARS-CoV-2 S protein comprising one or moremutations characteristic of an Omicron variant comprises a nucleotidesequence that is at least 80% identical (e.g., at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99%identical) to SEQ ID NO: 70. In some embodiments, a second RNA moleculeencoding a SARS-COV-2 S protein comprising one or more mutationscharacteristic of an Omicron variant comprises a nucleotide sequencethat is at least 80% identical to (e.g., at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical to) SEQID NO: 72. In some embodiments, a second RNA molecule encoding aSARS-COV-2 S protein comprising one or more mutations characteristic ofan Omicron variant comprises a nucleotide sequence that encodes an aminoacid sequence that is at least 80% identical to (e.g., at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to) SEQ ID NO: 69.

In some embodiments, a second RNA molecule encoding a SARS-CoV-2 Sprotein having one or more mutations that are characteristic of anOmicron variant comprises a nucleotide sequence that encodes the aminoacid sequence of SEQ ID NO: 74. In some embodiments, a second RNAmolecule encoding a SARS-CoV-2 S protein comprising one or moremutations characteristic of an Omicron variant comprises a nucleotidesequence that is at least 80% identical (e.g., at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99%identical) to SEQ ID NO: 75. In some embodiments, a second RNA moleculeencoding a SARS-COV-2 S protein comprising one or more mutationscharacteristic of an Omicron variant comprises a nucleotide sequencethat is at least 80% identical to (e.g., at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical to) SEQID NO: 77. In some embodiments, a second RNA molecule encoding aSARS-COV-2 S protein comprising one or more mutations characteristic ofan Omicron variant comprises a nucleotide sequence that encodes an aminoacid sequence that is at least 80% identical to (e.g., at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to) SEQ ID NO: 74.

In some embodiments, a nucleic acid containing particle (e.g., in someembodiments an LNP as described herein) comprises: a first RNA moleculecomprising a nucleotide sequence that encodes the amino acid sequence ofSEQ ID NO: 7 or an amino acid sequence that is at least 80% (e.g., atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or higher) identical to SEQ ID NO: 7); and a second RNAmolecule comprising a nucleotide sequence that encodes the amino acidsequence of SEQ ID NO: 49 or an amino acid sequence that is at least 80%(e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or higher) identical to SEQ ID NO: 49. In someembodiments, the ratio of the first RNA molecule that encodes the aminoacid sequence of SEQ ID NO: 7 or a sequence that is at least 80%identical to SEQ ID NO: 7 to the second RNA molecule that encodes theamino acid sequence of SEQ ID NO: 49 or a sequence that is at least 80%identical to SEQ ID NO: 49 is 1:1. In some embodiments, the ratio of thefirst RNA molecule that encodes the amino acid sequence of SEQ ID NO: 7or a sequence that is at least 80% identical to SEQ ID NO: 7 to thesecond RNA molecule that encodes the amino acid sequence of SEQ ID NO:49 or a sequence that is at least 80% identical to SEQ ID NO: 49 is 1:2.In some embodiments, the ratio of the first RNA molecule that encodesthe amino acid sequence of SEQ ID NO: 7 or a sequence that is at least80% identical to SEQ ID NO: 7 to the second RNA molecule that encodesthe amino acid sequence of SEQ ID NO: 49 or a sequence that is at least80% identical to SEQ ID NO: 49 is 1:3.

In some embodiments, a nucleic acid containing particle (e.g., in someembodiments an LNP as described herein) comprises: a first RNA moleculecomprising a nucleotide sequence of SEQ ID NO: 9 or a nucleotidesequence that is at least 80% (e.g., at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or higher) identicalto SEQ ID NO: 9); and a second RNA molecule comprising a nucleotidesequence of SEQ ID NO: 50 or a nucleotide sequence that is at least 80%(e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or higher) identical to SEQ ID NO: 50. In someembodiments, the ratio of the first RNA molecule comprising thenucleotide sequence of SEQ ID NO: 9 or a sequence that is at least 80%identical to SEQ ID NO: 9 to the second RNA molecule that comprises anucleotide sequence of SEQ ID NO: 50 or a sequence that is at least 80%identical to SEQ ID NO: 50 is 1:1. In some embodiments, the ratio of thefirst RNA molecule comprising the nucleotide sequence of SEQ ID NO: 9 ora sequence that is at least 80% identical to SEQ ID NO: 9 to the secondRNA molecule that comprises a nucleotide sequence of SEQ ID NO: 50 or asequence that is at least 80% identical to SEQ ID NO: 50 is 1:2. In someembodiments, the ratio of the first RNA molecule comprising thenucleotide sequence of SEQ ID NO: 9 or a sequence that is at least 80%identical to SEQ ID NO: 9 to the second RNA molecule that comprises anucleotide sequence of SEQ ID NO: 50 or a sequence that is at least 80%identical to SEQ ID NO: 50 is 1:3.

In some embodiments, a nucleic acid containing particle (e.g., in someembodiments an LNP as described herein) comprises: a first RNA moleculecomprising a nucleotide sequence of SEQ ID NO: 20 or a nucleotidesequence that is at least 80% (e.g., at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or higher) identicalto SEQ ID NO: 20; and a second RNA molecule comprising a nucleotidesequence of SEQ ID NO: 51 or a nucleotide sequence that is at least 80%(e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or higher) identical to SEQ ID NO: 51. In someembodiments, the ratio of the first RNA molecule comprising thenucleotide sequence of SEQ ID NO: 20 or a sequence that is at least 80%identical to SEQ ID NO: 20 to the second RNA molecule that comprises anucleotide sequence of SEQ ID NO: 51 or a sequence that is at least 80%identical to SEQ ID NO: 51 is 1:1. In some embodiments, the ratio of thefirst RNA molecule comprising the nucleotide sequence of SEQ ID NO: 20or a sequence that is at least 80% identical to SEQ ID NO: 20 to thesecond RNA molecule that comprises a nucleotide sequence of SEQ ID NO:51 or a sequence that is at least 80% identical to SEQ ID NO: 51 is 1:2.In some embodiments, the ratio of the first RNA molecule comprising thenucleotide sequence of SEQ ID NO: 20 or a sequence that is at least 80%identical to SEQ ID NO: 20 to the second RNA molecule that comprises anucleotide sequence of SEQ ID NO: 51 or a sequence that is at least 80%identical to SEQ ID NO: 51 is 1:3.

In some embodiments, a nucleic acid containing particle (e.g., in someembodiments an LNP as described herein) comprises: a first RNA moleculecomprising a nucleotide sequence that encodes the amino acid sequence ofSEQ ID NO: 7 or an amino acid sequence that is at least 80% (e.g., atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or higher) identical to SEQ ID NO: 7); and a second RNAmolecule comprising a nucleotide sequence that encodes the amino acidsequence of SEQ ID NO: 64 or an amino acid sequence that is at least 80%(e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or higher) identical to SEQ ID NO: 64. In someembodiments, the ratio of the first RNA molecule that encodes the aminoacid sequence of SEQ ID NO: 7 or a sequence that is at least 80%identical to SEQ ID NO: 7 to the second RNA molecule that encodes theamino acid sequence of SEQ ID NO: 49 or a sequence that is at least 80%identical to SEQ ID NO: 64 is 1:1. In some embodiments, the ratio of thefirst RNA molecule that encodes the amino acid sequence of SEQ ID NO: 7or a sequence that is at least 80% identical to SEQ ID NO: 7 to thesecond RNA molecule that encodes the amino acid sequence of SEQ ID NO:64 or a sequence that is at least 80% identical to SEQ ID NO: 64 is 1:2.In some embodiments, the ratio of the first RNA molecule that encodesthe amino acid sequence of SEQ ID NO: 7 or a sequence that is at least80% identical to SEQ ID NO: 7 to the second RNA molecule that encodesthe amino acid sequence of SEQ ID NO: 64 or a sequence that is at least80% identical to SEQ ID NO: 64 is 1:3.

In some embodiments, a nucleic acid containing particle (e.g., in someembodiments an LNP as described herein) comprises: a first RNA moleculecomprising a nucleotide sequence of SEQ ID NO: 9 or a nucleotidesequence that is at least 80% (e.g., at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or higher) identicalto SEQ ID NO: 9); and a second RNA molecule comprising a nucleotidesequence of SEQ ID NO: 65 or a nucleotide sequence that is at least 80%(e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or higher) identical to SEQ ID NO: 65. In someembodiments, the ratio of the first RNA molecule comprising thenucleotide sequence of SEQ ID NO: 9 or a sequence that is at least 80%identical to SEQ ID NO: 9 to the second RNA molecule that comprises anucleotide sequence of SEQ ID NO: 65 or a sequence that is at least 80%identical to SEQ ID NO: 65 is 1:1. In some embodiments, the ratio of thefirst RNA molecule comprising the nucleotide sequence of SEQ ID NO: 9 ora sequence that is at least 80% identical to SEQ ID NO: 9 to the secondRNA molecule that comprises a nucleotide sequence of SEQ ID NO: 65 or asequence that is at least 80% identical to SEQ ID NO: 65 is 1:2. In someembodiments, the ratio of the first RNA molecule comprising thenucleotide sequence of SEQ ID NO: 9 or a sequence that is at least 80%identical to SEQ ID NO: 9 to the second RNA molecule that comprises anucleotide sequence of SEQ ID NO: 65 or a sequence that is at least 80%identical to SEQ ID NO: 65 is 1:3.

In some embodiments, a nucleic acid containing particle (e.g., in someembodiments an LNP as described herein) comprises: a first RNA moleculecomprising a nucleotide sequence of SEQ ID NO: 20 or a nucleotidesequence that is at least 80% (e.g., at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or higher) identicalto SEQ ID NO: 20; and a second RNA molecule comprising a nucleotidesequence of SEQ ID NO: 51 or a nucleotide sequence that is at least 80%(e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or higher) identical to SEQ ID NO: 67. In someembodiments, the ratio of the first RNA molecule comprising thenucleotide sequence of SEQ ID NO: 20 or a sequence that is at least 80%identical to SEQ ID NO: 20 to the second RNA molecule that comprises anucleotide sequence of SEQ ID NO: 67 or a sequence that is at least 80%identical to SEQ ID NO: 67 is 1:1. In some embodiments, the ratio of thefirst RNA molecule comprising the nucleotide sequence of SEQ ID NO: 20or a sequence that is at least 80% identical to SEQ ID NO: 20 to thesecond RNA molecule that comprises a nucleotide sequence of SEQ ID NO:67 or a sequence that is at least 80% identical to SEQ ID NO: 67 is 1:2.In some embodiments, the ratio of the first RNA molecule comprising thenucleotide sequence of SEQ ID NO: 20 or a sequence that is at least 80%identical to SEQ ID NO: 20 to the second RNA molecule that comprises anucleotide sequence of SEQ ID NO: 67 or a sequence that is at least 80%identical to SEQ ID NO: 67 is 1:3.

In some embodiments, a nucleic acid containing particle (e.g., in someembodiments an LNP as described herein) comprises: a first RNA moleculecomprising a nucleotide sequence that encodes the amino acid sequence ofSEQ ID NO: 7 or an amino acid sequence that is at least 80% (e.g., atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or higher) identical to SEQ ID NO: 7); and a second RNAmolecule comprising a nucleotide sequence that encodes the amino acidsequence of SEQ ID NO: 69 or an amino acid sequence that is at least 80%(e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or higher) identical to SEQ ID NO: 69. In someembodiments, the ratio of the first RNA molecule that encodes the aminoacid sequence of SEQ ID NO: 7 or a sequence that is at least 80%identical to SEQ ID NO: 7 to the second RNA molecule that encodes theamino acid sequence of SEQ ID NO: 69 or a sequence that is at least 80%identical to SEQ ID NO: 69 is 1:1. In some embodiments, the ratio of thefirst RNA molecule that encodes the amino acid sequence of SEQ ID NO: 7or a sequence that is at least 80% identical to SEQ ID NO: 7 to thesecond RNA molecule that encodes the amino acid sequence of SEQ ID NO:69 or a sequence that is at least 80% identical to SEQ ID NO: 69 is 1:2.In some embodiments, the ratio of the first RNA molecule that encodesthe amino acid sequence of SEQ ID NO: 7 or a sequence that is at least80% identical to SEQ ID NO: 7 to the second RNA molecule that encodesthe amino acid sequence of SEQ ID NO: 69 or a sequence that is at least80% identical to SEQ ID NO: 69 is 1:3.

In some embodiments, a nucleic acid containing particle (e.g., in someembodiments an LNP as described herein) comprises: a first RNA moleculecomprising a nucleotide sequence of SEQ ID NO: 9 or a nucleotidesequence that is at least 80% (e.g., at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or higher) identicalto SEQ ID NO: 9); and a second RNA molecule comprising a nucleotidesequence of SEQ ID NO: 70 or a nucleotide sequence that is at least 80%(e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or higher) identical to SEQ ID NO: 70. In someembodiments, the ratio of the first RNA molecule comprising thenucleotide sequence of SEQ ID NO: 9 or a sequence that is at least 80%identical to SEQ ID NO: 9 to the second RNA molecule that comprises anucleotide sequence of SEQ ID NO: 70 or a sequence that is at least 80%identical to SEQ ID NO: 70 is 1:1. In some embodiments, the ratio of thefirst RNA molecule comprising the nucleotide sequence of SEQ ID NO: 9 ora sequence that is at least 80% identical to SEQ ID NO: 9 to the secondRNA molecule that comprises a nucleotide sequence of SEQ ID NO: 70 or asequence that is at least 80% identical to SEQ ID NO: 70 is 1:2. In someembodiments, the ratio of the first RNA molecule comprising thenucleotide sequence of SEQ ID NO: 9 or a sequence that is at least 80%identical to SEQ ID NO: 9 to the second RNA molecule that comprises anucleotide sequence of SEQ ID NO: 50 or a sequence that is at least 80%identical to SEQ ID NO: 70 is 1:3.

In some embodiments, a nucleic acid containing particle (e.g., in someembodiments an LNP as described herein) comprises: a first RNA moleculecomprising a nucleotide sequence of SEQ ID NO: 20 or a nucleotidesequence that is at least 80% (e.g., at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or higher) identicalto SEQ ID NO: 20; and a second RNA molecule comprising a nucleotidesequence of SEQ ID NO: 72 or a nucleotide sequence that is at least 80%(e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or higher) identical to SEQ ID NO: 72. In someembodiments, the ratio of the first RNA molecule comprising thenucleotide sequence of SEQ ID NO: 20 or a sequence that is at least 80%identical to SEQ ID NO: 20 to the second RNA molecule that comprises anucleotide sequence of SEQ ID NO: 72 or a sequence that is at least 80%identical to SEQ ID NO: 72 is 1:1. In some embodiments, the ratio of thefirst RNA molecule comprising the nucleotide sequence of SEQ ID NO: 20or a sequence that is at least 80% identical to SEQ ID NO: 20 to thesecond RNA molecule that comprises a nucleotide sequence of SEQ ID NO:72 or a sequence that is at least 80% identical to SEQ ID NO: 72 is 1:2.In some embodiments, the ratio of the first RNA molecule comprising thenucleotide sequence of SEQ ID NO: 20 or a sequence that is at least 80%identical to SEQ ID NO: 20 to the second RNA molecule that comprises anucleotide sequence of SEQ ID NO: 72 or a sequence that is at least 80%identical to SEQ ID NO: 72 is 1:3.

In some embodiments, a nucleic acid containing particle (e.g., in someembodiments an LNP as described herein) comprises: a first RNA moleculecomprising a nucleotide sequence that encodes the amino acid sequence ofSEQ ID NO: 7 or an amino acid sequence that is at least 80% (e.g., atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or higher) identical to SEQ ID NO: 7); and a second RNAmolecule comprising a nucleotide sequence that encodes the amino acidsequence of SEQ ID NO: 74 or an amino acid sequence that is at least 80%(e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or higher) identical to SEQ ID NO: 74. In someembodiments, the ratio of the first RNA molecule that encodes the aminoacid sequence of SEQ ID NO: 7 or a sequence that is at least 80%identical to SEQ ID NO: 7 to the second RNA molecule that encodes theamino acid sequence of SEQ ID NO: 74 or a sequence that is at least 80%identical to SEQ ID NO: 74 is 1:1. In some embodiments, the ratio of thefirst RNA molecule that encodes the amino acid sequence of SEQ ID NO: 7or a sequence that is at least 80% identical to SEQ ID NO: 7 to thesecond RNA molecule that encodes the amino acid sequence of SEQ ID NO:74 or a sequence that is at least 80% identical to SEQ ID NO: 74 is 1:2.In some embodiments, the ratio of the first RNA molecule that encodesthe amino acid sequence of SEQ ID NO: 7 or a sequence that is at least80% identical to SEQ ID NO: 7 to the second RNA molecule that encodesthe amino acid sequence of SEQ ID NO: 74 or a sequence that is at least80% identical to SEQ ID NO: 74 is 1:3.

In some embodiments, a nucleic acid containing particle (e.g., in someembodiments an LNP as described herein) comprises: a first RNA moleculecomprising a nucleotide sequence of SEQ ID NO: 9 or a nucleotidesequence that is at least 80% (e.g., at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or higher) identicalto SEQ ID NO: 9); and a second RNA molecule comprising a nucleotidesequence of SEQ ID NO: 75 or a nucleotide sequence that is at least 80%(e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or higher) identical to SEQ ID NO: 75. In someembodiments, the ratio of the first RNA molecule comprising thenucleotide sequence of SEQ ID NO: 9 or a sequence that is at least 80%identical to SEQ ID NO: 9 to the second RNA molecule that comprises anucleotide sequence of SEQ ID NO: 75 or a sequence that is at least 80%identical to SEQ ID NO: 75 is 1:1. In some embodiments, the ratio of thefirst RNA molecule comprising the nucleotide sequence of SEQ ID NO: 9 ora sequence that is at least 80% identical to SEQ ID NO: 9 to the secondRNA molecule that comprises a nucleotide sequence of SEQ ID NO: 75 or asequence that is at least 80% identical to SEQ ID NO: 75 is 1:2. In someembodiments, the ratio of the first RNA molecule comprising thenucleotide sequence of SEQ ID NO: 9 or a sequence that is at least 80%identical to SEQ ID NO: 9 to the second RNA molecule that comprises anucleotide sequence of SEQ ID NO: 75 or a sequence that is at least 80%identical to SEQ ID NO: 75 is 1:3.

In some embodiments, a nucleic acid containing particle (e.g., in someembodiments an LNP as described herein) comprises: a first RNA moleculecomprising a nucleotide sequence of SEQ ID NO: 20 or a nucleotidesequence that is at least 80% (e.g., at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or higher) identicalto SEQ ID NO: 20; and a second RNA molecule comprising a nucleotidesequence of SEQ ID NO: 77 or a nucleotide sequence that is at least 80%(e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or higher) identical to SEQ ID NO: 77. In someembodiments, the ratio of the first RNA molecule comprising thenucleotide sequence of SEQ ID NO: 20 or a sequence that is at least 80%identical to SEQ ID NO: 20 to the second RNA molecule that comprises anucleotide sequence of SEQ ID NO: 77 or a sequence that is at least 80%identical to SEQ ID NO: 77 is 1:1. In some embodiments, the ratio of thefirst RNA molecule comprising the nucleotide sequence of SEQ ID NO: 20or a sequence that is at least 80% identical to SEQ ID NO: 20 to thesecond RNA molecule that comprises a nucleotide sequence of SEQ ID NO:77 or a sequence that is at least 80% identical to SEQ ID NO: 77 is 1:2.In some embodiments, the ratio of the first RNA molecule comprising thenucleotide sequence of SEQ ID NO: 20 or a sequence that is at least 80%identical to SEQ ID NO: 20 to the second RNA molecule that comprises anucleotide sequence of SEQ ID NO: 77 or a sequence that is at least 80%identical to SEQ ID NO: 77 is 1:3.

In some embodiments, a nucleic acid containing particle (e.g., in someembodiments an LNP as described herein) comprises: a first RNA moleculecomprising a nucleotide sequence that encodes the amino acid sequence ofSEQ ID NO: 7 or an amino acid sequence that is at least 80% (e.g., atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or higher) identical to SEQ ID NO: 7); and a second RNAmolecule comprising a nucleotide sequence that encodes the amino acidsequence of SEQ ID NO: 55, 58, or 61 or an amino acid sequence that isat least 80% (e.g., at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99% or higher) identical to SEQ ID NO: 55,58, or 61.

In some embodiments, a nucleic acid containing particle (e.g., in someembodiments an LNP as described herein) comprises: a first RNA moleculecomprising a nucleotide sequence of SEQ ID NO: 9 or a nucleotidesequence that is at least 80% (e.g., at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or higher) identicalto SEQ ID NO: 9; and a second RNA molecule comprising a nucleotidesequence of SEQ ID NO: 56, 59, or 62a or a nucleotide sequence that isat least 80% (e.g., at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99% or higher) identical to SEQ ID NO: 56,59, or 62a.

In some embodiments, a nucleic acid containing particle (e.g., in someembodiments an LNP as described herein) comprises: a first RNA moleculecomprising a nucleotide sequence of SEQ ID NO: 20 or a nucleotidesequence that is at least 80% (e.g., at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or higher) identicalto SEQ ID NO: 20; and a second RNA molecule comprising a nucleotidesequence of SEQ ID NO: 57, 60, or 63a or a nucleotide sequence that isat least 80% (e.g., at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99% or higher) identical to SEQ ID NO: 57,60, or 63a.

In some embodiments, a nucleic acid containing particle (e.g., in someembodiments an LNP as described herein) comprises: a first RNA moleculecomprising a nucleotide sequence that encodes the amino acid sequence ofSEQ ID NO: 58 or an amino acid sequence that is at least 80% (e.g., atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or higher) identical to SEQ ID NO: 58; and a second RNAmolecule comprising a nucleotide sequence that encodes an amino acidsequence of SEQ ID NO: 49, 55, or 61 or an amino acid sequence that isat least 80% (e.g., at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99% or higher) identical to SEQ ID NO: 49,55, or 61.

In some embodiments, a nucleic acid containing particle (e.g., in someembodiments an LNP as described herein) comprises: a first RNA moleculecomprising a nucleotide sequence of SEQ ID NO: 59 or a nucleotidesequence that is at least 80% (e.g., at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or higher) identicalto SEQ ID NO: 59; and a second RNA molecule comprising a nucleotidesequence of SEQ ID NO: 50, 56, or 62a, or a nucleotide sequence that isat least 80% (e.g., at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99% or higher) identical to SEQ ID NO: 50,56, or 62a.

In some embodiments, a nucleic acid containing particle (e.g., in someembodiments an LNP as described herein) comprises: a first RNA moleculecomprising a nucleotide sequence of SEQ ID NO: 60 or a nucleotidesequence that is at least 80% (e.g., at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or higher) identicalto SEQ ID NO: 60; and a second RNA molecule comprising a nucleotidesequence of SEQ ID NO: 51, 57, or 63a, or a nucleotide sequence that isat least 80% (e.g., at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99% or higher) identical to SEQ ID NO: 51,57, or 63a.

In some embodiments, a nucleic acid containing particle (e.g., in someembodiments an LNP as described herein) comprises: a first RNA moleculecomprising a nucleotide sequence that encodes the amino acid sequence ofSEQ ID NO: 49 or an amino acid sequence that is at least 80% (e.g., atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or higher) identical to SEQ ID NO: 49; and a second RNAmolecule comprising a nucleotide sequence that encodes the amino acidsequence of SEQ ID NO: 55 or 61 or an amino acid sequence that is atleast 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%,at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or higher) identical to SEQ ID NO: 55 or 61. Insome embodiments, a nucleic acid containing particle (e.g., in someembodiments an LNP as described herein) comprises: a first RNA moleculecomprising a nucleotide sequence of SEQ ID NO: 50 or a nucleotidesequence that is at least 80% (e.g., at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or higher) identicalto SEQ ID NO: 50; and a second RNA molecule comprising a nucleotidesequence of SEQ ID NO: 56 or 62a or a nucleotide sequence that is atleast 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%,at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or higher) identical to SEQ ID NO: 56 or 62a.

In some embodiments, a nucleic acid containing particle (e.g., in someembodiments an LNP as described herein) comprises: a first RNA moleculecomprising a nucleotide sequence of SEQ ID NO: 51 or a nucleotidesequence that is at least 80% (e.g., at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or higher) identicalto SEQ ID NO: 51; and a second RNA molecule comprising a nucleotidesequence of SEQ ID NO: 57 or 63a or a nucleotide sequence that is atleast 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%,at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or higher) identical to SEQ ID NO: 57 or 63a.

In some embodiments, a nucleic acid containing particle (e.g., in someembodiments an LNP as described herein) comprises: a first RNA moleculecomprising a nucleotide sequence that encodes the amino acid sequence ofSEQ ID NO: 55 or an amino acid sequence that is at least 80% (e.g., atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or higher) identical to SEQ ID NO: 55; and a second RNAmolecule comprising a nucleotide sequence that encodes the amino acidsequence of SEQ ID NO: 61 or an amino acid sequence that is at least 80%(e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or higher) identical to SEQ ID NO: 61.

In some embodiments, a nucleic acid containing particle (e.g., in someembodiments an LNP as described herein) comprises: a first RNA moleculecomprising a nucleotide sequence of SEQ ID NO: 56 or a nucleotidesequence that is at least 80% (e.g., at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or higher) identicalto SEQ ID NO: 56; and a second RNA molecule comprising a nucleotidesequence of SEQ ID NO: 62a, or a nucleotide sequence that is at least80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or higher) identical to SEQ ID NO: 62a. In someembodiments, a nucleic acid containing particle (e.g., in someembodiments an LNP as described herein) comprises: a first RNA moleculecomprising a nucleotide sequence of SEQ ID NO: 57 or a nucleotidesequence that is at least 80% (e.g., at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or higher) identicalto SEQ ID NO: 57; and a second RNA molecule comprising a nucleotidesequence of SEQ ID NO: 63a or a nucleotide sequence that is at least 80%(e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or higher) identical to SEQ ID NO: 63a.

In some embodiments, a particle (e.g., in some embodiments an LNP)containing nucleic acids (e.g., RNAs) encoding different polypeptidescan be formed by mixing a plurality of (e.g., at least two, at leastthree, or more) RNA molecules with particle-forming components (e.g.,lipids). In some embodiments, nucleic acids (e.g., RNAs) encodingdifferent polypeptides can be mixed (e.g., in some embodiments insubstantially equal proportions, e.g., in some embodiments at a 1:1ratio when two RNA molecules are present) prior to mixing withparticle-forming components (e.g., lipids).

In some embodiments, two or more RNA molecules each encoding a differentpolypeptide (e.g., as described herein) can be mixed withparticle-forming agents to form nucleic acid containing particles asdescribed above. In alternative embodiments, two or more RNA moleculeseach encoding a different polypeptide (e.g., as described herein) can beformulated into separate particle compositions, which are then mixedtogether. For example, in some embodiments, individual populations ofnucleic acid containing particles, each population comprising an RNAmolecule encoding a different immunogenic polypeptide or immunogenicfragment thereof (e.g., as described herein), can be separately formedand then mixed together, for example, prior to filling into vials duringa manufacturing process, or immediately prior to administration (e.g.,by an administering health-care professional)). Accordingly, in someembodiments, described herein is a composition comprises two or morepopulations of particles (e.g., in some embodiments, lipidnanoparticles), each population comprising at least one RNA moleculeencoding a different immunogenic polypeptide or immunogenic fragmentthereof (e.g., a SARS-CoV-2 S protein, or fragments thereof, from adifferent variant). In some embodiments, each population may be providedin a composition at a desirable proportion (e.g., in some embodiments,each population may be provided in a composition in an amount thatprovides the same amount of RNA molecules).

Cationic Polymer

Given their high degree of chemical flexibility, polymers are commonlyused materials for nanoparticle-based delivery. Typically, cationicpolymers are used to electrostatically condense the negatively chargednucleic acid into nanoparticles. These positively charged groups oftenconsist of amines that change their state of protonation in the pH rangebetween 5.5 and 7.5, thought to lead to an ion imbalance that results inendosomal rupture. Polymers such as poly-L-lysine, polyamidoamine,protamine and polyethyleneimine, as well as naturally occurring polymerssuch as chitosan have all been applied to nucleic acid delivery and aresuitable as cationic polymers herein. In addition, some investigatorshave synthesized polymers specifically for nucleic acid delivery.Poly(R-amino esters), in particular, have gained widespread use innucleic acid delivery owing to their ease of synthesis andbiodegradability. Such synthetic polymers are also suitable as cationicpolymers herein.

A “polymer,” as used herein, is given its ordinary meaning, i.e., amolecular structure comprising one or more repeat units (monomers),connected by covalent bonds. The repeat units can all be identical, orin some cases, there can be more than one type of repeat unit presentwithin the polymer. In some cases, the polymer is biologically derived,i.e., a biopolymer such as a protein. In some cases, additional moietiescan also be present in the polymer, for example targeting moieties suchas those described herein.

If more than one type of repeat unit is present within the polymer, thenthe polymer is said to be a “copolymer.” It is to be understood that thepolymer being employed herein can be a copolymer. The repeat unitsforming the copolymer can be arranged in any fashion. For example, therepeat units can be arranged in a random order, in an alternating order,or as a “block” copolymer, i.e., comprising one or more regions eachcomprising a first repeat unit (e.g., a first block), and one or moreregions each comprising a second repeat unit (e.g., a second block),etc. Block copolymers can have two (a diblock copolymer), three (atriblock copolymer), or more numbers of distinct blocks.

In certain embodiments, the polymer is biocompatible. Biocompatiblepolymers are polymers that typically do not result in significant celldeath at moderate concentrations. In certain embodiments, thebiocompatible polymer is biodegradable, i.e., the polymer is able todegrade, chemically and/or biologically, within a physiologicalenvironment, such as within the body.

In certain embodiments, polymer may be protamine or polyalkyleneimine,in particular protamine.

The term “protamine” refers to any of various strongly basic proteins ofrelatively low molecular weight that are rich in arginine and are foundassociated especially with DNA in place of somatic histones in the spermcells of various animals (as fish). In particular, the term “protamine”refers to proteins found in fish sperm that are strongly basic, aresoluble in water, are not coagulated by heat, and yield chiefly arginineupon hydrolysis. In purified form, they are used in a long-actingformulation of insulin and to neutralize the anticoagulant effects ofheparin.

According to the present disclosure, the term “protamine” as used hereinis meant to comprise any protamine amino acid sequence obtained orderived from natural or biological sources including fragments thereofand multimeric forms of said amino acid sequence or fragment thereof aswell as (synthesized) polypeptides which are artificial and specificallydesigned for specific purposes and cannot be isolated from native orbiological sources.

In one embodiment, the polyalkyleneimine comprises polyethylenimineand/or polypropylenimine, preferably polyethyleneimine. A preferredpolyalkyleneimine is polyethyleneimine (PEI). The average molecularweight of PEI is preferably 0.75·10² to 10⁷ Da, preferably 1000 to 10⁵Da, more preferably 10000 to 40000 Da, more preferably 15000 to 30000Da, even more preferably 20000 to 25000 Da.

Preferred according to the present disclosure is linearpolyalkyleneimine such as linear polyethyleneimine (PEI).

Cationic polymers (including polycationic polymers) contemplated for useherein include any cationic polymers which are able to electrostaticallybind nucleic acid. In one embodiment, cationic polymers contemplated foruse herein include any cationic polymers with which nucleic acid can beassociated, e.g. by forming complexes with the nucleic acid or formingvesicles in which the nucleic acid is enclosed or encapsulated.

Particles described herein may also comprise polymers other thancationic polymers, i.e., non-cationic polymers and/or anionic polymers.Collectively, anionic and neutral polymers are referred to herein asnon-cationic polymers.

Lipid and Lipid-Like Material

The terms “lipid” and “lipid-like material” are broadly defined hereinas molecules which comprise one or more hydrophobic moieties or groupsand optionally also one or more hydrophilic moieties or groups.Molecules comprising hydrophobic moieties and hydrophilic moieties arealso frequently denoted as amphiphiles. Lipids are usually poorlysoluble in water. In an aqueous environment, the amphiphilic natureallows the molecules to self-assemble into organized structures anddifferent phases. One of those phases consists of lipid bilayers, asthey are present in vesicles, multilamellar/unilamellar liposomes, ormembranes in an aqueous environment. Hydrophobicity can be conferred bythe inclusion of apolar groups that include, but are not limited to,long-chain saturated and unsaturated aliphatic hydrocarbon groups andsuch groups substituted by one or more aromatic, cycloaliphatic, orheterocyclic group(s). The hydrophilic groups may comprise polar and/orcharged groups and include carbohydrates, phosphate, carboxylic,sulfate, amino, sulfhydryl, nitro, hydroxyl, and other like groups.

As used herein, the term “amphiphilic” refers to a molecule having botha polar portion and a non-polar portion. Often, an amphiphilic compoundhas a polar head attached to a long hydrophobic tail. In someembodiments, the polar portion is soluble in water, while the non-polarportion is insoluble in water. In addition, the polar portion may haveeither a formal positive charge, or a formal negative charge.Alternatively, the polar portion may have both a formal positive and anegative charge, and be a zwitterion or inner salt. For purposes of thepresent disclosure, the amphiphilic compound can be, but is not limitedto, one or a plurality of natural or non-natural lipids and lipid-likecompounds.

The term “lipid-like material”, “lipid-like compound” or “lipid-likemolecule” relates to substances that structurally and/or functionallyrelate to lipids but may not be considered as lipids in a strict sense.For example, the term includes compounds that are able to formamphiphilic layers as they are present in vesicles,multilamellar/unilamellar liposomes, or membranes in an aqueousenvironment and includes surfactants, or synthesized compounds with bothhydrophilic and hydrophobic moieties. Generally speaking, the termrefers to molecules, which comprise hydrophilic and hydrophobic moietieswith different structural organization, which may or may not be similarto that of lipids. As used herein, the term “lipid” is to be construedto cover both lipids and lipid-like materials unless otherwise indicatedherein or clearly contradicted by context.

Specific examples of amphiphilic compounds that may be included in anamphiphilic layer include, but are not limited to, phospholipids,aminolipids and sphingolipids.

In certain embodiments, the amphiphilic compound is a lipid. The term“lipid” refers to a group of organic compounds that are characterized bybeing insoluble in water, but soluble in many organic solvents.Generally, lipids may be divided into eight categories: fatty acids,glycerolipids, glycerophospholipids, sphingolipids, saccharolipids,polyketides (derived from condensation of ketoacyl subunits), sterollipids and prenol lipids (derived from condensation of isoprenesubunits). Although the term “lipid” is sometimes used as a synonym forfats, fats are a subgroup of lipids called triglycerides. Lipids alsoencompass molecules such as fatty acids and their derivatives (includingtri-, di-, monoglycerides, and phospholipids), as well assterol-containing metabolites such as cholesterol.

Fatty acids, or fatty acid residues are a diverse group of moleculesmade of a hydrocarbon chain that terminates with a carboxylic acidgroup; this arrangement confers the molecule with a polar, hydrophilicend, and a nonpolar, hydrophobic end that is insoluble in water. Thecarbon chain, typically between four and 24 carbons long, may besaturated or unsaturated, and may be attached to functional groupscontaining oxygen, halogens, nitrogen, and sulfur. If a fatty acidcontains a double bond, there is the possibility of either a cis ortrans geometric isomerism, which significantly affects the molecule'sconfiguration. Cis-double bonds cause the fatty acid chain to bend, aneffect that is compounded with more double bonds in the chain. Othermajor lipid classes in the fatty acid category are the fatty esters andfatty amides. Glycerolipids are composed of mono-, di-, andtri-substituted glycerols, the best-known being the fatty acid triestersof glycerol, called triglycerides. The word “triacylglycerol” issometimes used synonymously with “triglyceride”. In these compounds, thethree hydroxyl groups of glycerol are each esterified, typically bydifferent fatty acids. Additional subclasses of glycerolipids arerepresented by glycosylglycerols, which are characterized by thepresence of one or more sugar residues attached to glycerol via aglycosidic linkage.

The glycerophospholipids are amphipathic molecules (containing bothhydrophobic and hydrophilic regions) that contain a glycerol core linkedto two fatty acid-derived “tails” by ester linkages and to one “head”group by a phosphate ester linkage. Examples of glycerophospholipids,usually referred to as phospholipids (though sphingomyelins are alsoclassified as phospholipids) are phosphatidylcholine (also known as PC,GPCho or lecithin), phosphatidylethanolamine (PE or GPEtn) andphosphatidylserine (PS or GPSer).

Sphingolipids are a complex family of compounds that share a commonstructural feature, a sphingoid base backbone. The major sphingoid basein mammals is commonly referred to as sphingosine. Ceramides(N-acyl-sphingoid bases) are a major subclass of sphingoid basederivatives with an amide-linked fatty acid. The fatty acids aretypically saturated or mono-unsaturated with chain lengths from 16 to 26carbon atoms. The major phosphosphingolipids of mammals aresphingomyelins (ceramide phosphocholines), whereas insects containmainly ceramide phosphoethanolamines and fungi have phytoceramidephosphoinositols and mannose-containing headgroups. Theglycosphingolipids are a diverse family of molecules composed of one ormore sugar residues linked via a glycosidic bond to the sphingoid base.Examples of these are the simple and complex glycosphingolipids such ascerebrosides and gangliosides.

Sterol lipids, such as cholesterol and its derivatives, or tocopheroland its derivatives, are an important component of membrane lipids,along with the glycerophospholipids and sphingomyelins.

Saccharolipids describe compounds in which fatty acids are linkeddirectly to a sugar backbone, forming structures that are compatiblewith membrane bilayers. In the saccharolipids, a monosaccharidesubstitutes for the glycerol backbone present in glycerolipids andglycerophospholipids. The most familiar saccharolipids are the acylatedglucosamine precursors of the Lipid A component of thelipopolysaccharides in Gram-negative bacteria. Typical lipid A moleculesare disaccharides of glucosamine, which are derivatized with as many asseven fatty-acyl chains. The minimal lipopolysaccharide required forgrowth in E. coli is Kdo2-Lipid A, a hexa-acylated disaccharide ofglucosamine that is glycosylated with two 3-deoxy-D-manno-octulosonicacid (Kdo) residues.

Polyketides are synthesized by polymerization of acetyl and propionylsubunits by classic enzymes as well as iterative and multimodularenzymes that share mechanistic features with the fatty acid synthases.They comprise a large number of secondary metabolites and naturalproducts from animal, plant, bacterial, fungal and marine sources, andhave great structural diversity. Many polyketides are cyclic moleculeswhose backbones are often further modified by glycosylation,methylation, hydroxylation, oxidation, or other processes.

According to the present disclosure, lipids and lipid-like materials maybe cationic, anionic or neutral. Neutral lipids or lipid-like materialsexist in an uncharged or neutral zwitterionic form at a selected pH.

Cationic or Cationically Ionizable Lipids or Lipid-Like Materials

The nucleic acid particles described herein may comprise at least onecationic or cationically ionizable lipid or lipid-like material asparticle forming agent. Cationic or cationically ionizable lipids orlipid-like materials contemplated for use herein include any cationic orcationically ionizable lipids or lipid-like materials which are able toelectrostatically bind nucleic acid. In one embodiment, cationic orcationically ionizable lipids or lipid-like materials contemplated foruse herein can be associated with nucleic acid, e.g. by formingcomplexes with the nucleic acid or forming vesicles in which the nucleicacid is enclosed or encapsulated.

As used herein, a “cationic lipid” or “cationic lipid-like material”refers to a lipid or lipid-like material having a net positive charge.Cationic lipids or lipid-like materials bind negatively charged nucleicacid by electrostatic interaction. Generally, cationic lipids possess alipophilic moiety, such as a sterol, an acyl chain, a diacyl or moreacyl chains, and the head group of the lipid typically carries thepositive charge.

In certain embodiments, a cationic lipid or lipid-like material has anet positive charge only at certain pH, in particular acidic pH, whileit has preferably no net positive charge, preferably has no charge,i.e., it is neutral, at a different, preferably higher pH such asphysiological pH. This ionizable behavior is thought to enhance efficacythrough helping with endosomal escape and reducing toxicity as comparedwith particles that remain cationic at physiological pH. For purposes ofthe present disclosure, such “cationically ionizable” lipids orlipid-like materials are comprised by the term “cationic lipid orlipid-like material” unless contradicted by the circumstances.

In one embodiment, the cationic or cationically ionizable lipid orlipid-like material comprises a head group which includes at least onenitrogen atom (N) which is positive charged or capable of beingprotonated.

Examples of cationic lipids include, but are not limited to1,2-dioleoyl-3-trimethylammonium propane (DOTAP);N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA),1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA),3-(N-(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol),dimethyldioctadecylammonium (DDAB);1,2-dioleoyl-3-dimethylammonium-propane (DODAP);1,2-diacyloxy-3-dimethylammonium propanes;1,2-dialkyloxy-3-dimethylammonium propanes; dioctadecyldimethyl ammoniumchloride (DODAC), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA),2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium (DMRIE),1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC),1,2-dimyristoyl-3-trimethylammonium propane (DMTAP),1,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE),and 2,3-dioleoyloxy-N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-1-propanamium trifluoroacetate (DOSPA),1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),dioctadecylamidoglycyl spermine (DOGS),3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-oc-tadecadienoxy)propane(CLinDMA),2-[5′-(cholest-5-en-3-beta-oxy)-3′-oxapentoxy)-3-dimethyl-1-(cis,cis-9′,12′-octadecadienoxy)propane(CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA),1,2-N,N′-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP),2,3-Dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP),1,2-N,N′-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP),1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP),2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA),2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-XTC2-DMA),2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA),heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate(DLin-MC3-DMA),N-(2-Hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminiumbromide (DMRIE),(±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecenyloxy)-1-propanaminiumbromide (GAP-DMORIE),(±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminiumbromide (GAP-DLRIE),(±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminiumbromide (GAP-DMRIE),N-(2-Aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminiumbromide (PAE-DMRIE),N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium(DOBAQ),2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine(Octyl-CLinDMA), 1,2-dimyristoyl-3-dimethylammonium-propane (DMDAP),1,2-dipalmitoyl-3-dimethylammonium-propane (DPDAP),N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide(MVL5), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC),2,3-bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N-dimethylpropan-1-ammoniumbromide (DLRIE),N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-aminiumbromide (DMORIE), di((Z)-non-2-en-1-yl)8,8′-((((2(dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate(ATX), N,N-dimethyl-2,3-bis(dodecyloxy)propan-1-amine (DLDMA),N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-amine (DMDMA),Di((Z)-non-2-en-1-yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecanedioate(L319),N-Dodecyl-3-((2-dodecylcarbamoyl-ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl-ethyl)-[2-(2-dodecylcarbamoyl-ethylamino)-ethyl]-amino}-ethylamino)propionamide(lipidoid 98N₁₂-5),1-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2hydroxydodecyl)amino]ethyl]piperazin-1-yl]ethyl]amino]dodecan-2-ol(lipidoid C12-200).

In some embodiments, the cationic lipid may comprise from about 10 mol %to about 100 mol %, about 20 mol % to about 100 mol %, about 30 mol % toabout 100 mol %, about 40 mol % to about 100 mol %, or about 50 mol % toabout 100 mol % of the total lipid present in the particle.

Additional Lipids or Lipid-Like Materials

Particles described herein may also comprise lipids or lipid-likematerials other than cationic or cationically ionizable lipids orlipid-like materials, i.e., non-cationic lipids or lipid-like materials(including non-cationically ionizable lipids or lipid-like materials).Collectively, anionic and neutral lipids or lipid-like materials arereferred to herein as non-cationic lipids or lipid-like materials.Optimizing the formulation of nucleic acid particles by addition ofother hydrophobic moieties, such as cholesterol and lipids, in additionto an ionizable/cationic lipid or lipid-like material may enhanceparticle stability and efficacy of nucleic acid delivery.

An additional lipid or lipid-like material may be incorporated which mayor may not affect the overall charge of the nucleic acid particles. Incertain embodiments, the additional lipid or lipid-like material is anon-cationic lipid or lipid-like material. The non-cationic lipid maycomprise, e.g., one or more anionic lipids and/or neutral lipids. Asused herein, an “anionic lipid” refers to any lipid that is negativelycharged at a selected pH. As used herein, a “neutral lipid” refers toany of a number of lipid species that exist either in an uncharged orneutral zwitterionic form at a selected pH. In preferred embodiments,the additional lipid comprises one of the following neutral lipidcomponents: (1) a phospholipid, (2) cholesterol or a derivative thereof;or (3) a mixture of a phospholipid and cholesterol or a derivativethereof. Examples of cholesterol derivatives include, but are notlimited to, cholestanol, cholestanone, cholestenone, coprostanol,cholesteryl-2′-hydroxyethyl ether, cholesteryl-4′-hydroxybutyl ether,tocopherol and derivatives thereof, and mixtures thereof.

Specific phospholipids that can be used include, but are not limited to,phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols,phosphatidic acids, phosphatidylserines or sphingomyelin. Suchphospholipids include in particular diacylphosphatidylcholines, such asdistearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine(DOPC), dimyristoylphosphatidylcholine (DMPC),dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine,dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine(DAPC), dibehenoylphosphatidylcholine (DBPC),ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine(DLPC), palmitoyloleoyl-phosphatidylcholine (POPC),1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC),1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine(OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC) andphosphatidylethanolamines, in particulardiacylphosphatidylethanolamines, such asdioleoylphosphatidylethanolamine (DOPE),distearoyl-phosphatidylethanolamine (DSPE),dipalmitoyl-phosphatidylethanolamine (DPPE),dimyristoyl-phosphatidylethanolamine (DMPE),dilauroyl-phosphatidylethanolamine (DLPE),diphytanoyl-phosphatidylethanolamine (DPyPE), and furtherphosphatidylethanolamine lipids with different hydrophobic chains.

In certain preferred embodiments, the additional lipid is DSPC or DSPCand cholesterol.

In certain embodiments, the nucleic acid particles include both acationic lipid and an additional lipid.

In one embodiment, particles described herein include a polymerconjugated lipid such as a pegylated lipid. The term “pegylated lipid”refers to a molecule comprising both a lipid portion and a polyethyleneglycol portion. Pegylated lipids are known in the art.

Without wishing to be bound by theory, the amount of the at least onecationic lipid compared to the amount of the at least one additionallipid may affect important nucleic acid particle characteristics, suchas charge, particle size, stability, tissue selectivity, and bioactivityof the nucleic acid. Accordingly, in some embodiments, the molar ratioof the at least one cationic lipid to the at least one additional lipidis from about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 toabout 1:1.

In some embodiments, the non-cationic lipid, in particular neutrallipid, (e.g., one or more phospholipids and/or cholesterol) may comprisefrom about 0 mol % to about 90 mol %, from about 0 mol % to about 80 mol%, from about 0 mol % to about 70 mol %, from about 0 mol % to about 60mol %, or from about 0 mol % to about 50 mol %, of the total lipidpresent in the particle.

Lipoplex Particles

In certain embodiments of the present disclosure, the RNA describedherein may be present in RNA lipoplex particles.

In the context of the present disclosure, the term “RNA lipoplexparticle” relates to a particle that contains lipid, in particularcationic lipid, and RNA. Electrostatic interactions between positivelycharged liposomes and negatively charged RNA results in complexation andspontaneous formation of RNA lipoplex particles. Positively chargedliposomes may be generally synthesized using a cationic lipid, such asDOTMA, and additional lipids, such as DOPE. In one embodiment, a RNAlipoplex particle is a nanoparticle.

In certain embodiments, the RNA lipoplex particles include both acationic lipid and an additional lipid. In an exemplary embodiment, thecationic lipid is DOTMA and the additional lipid is DOPE.

In some embodiments, the molar ratio of the at least one cationic lipidto the at least one additional lipid is from about 10:0 to about 1:9,about 4:1 to about 1:2, or about 3:1 to about 1:1. In specificembodiments, the molar ratio may be about 3:1, about 2.75:1, about2.5:1, about 2.25:1, about 2:1, about 1.75:1, about 1.5:1, about 1.25:1,or about 1:1. In an exemplary embodiment, the molar ratio of the atleast one cationic lipid to the at least one additional lipid is about2:1.

RNA lipoplex particles described herein have an average diameter that inone embodiment ranges from about 200 nm to about 1000 nm, from about 200nm to about 800 nm, from about 250 to about 700 nm, from about 400 toabout 600 nm, from about 300 nm to about 500 nm, or from about 350 nm toabout 400 nm. In specific embodiments, the RNA lipoplex particles havean average diameter of about 200 nm, about 225 nm, about 250 nm, about275 nm, about 300 nm, about 325 nm, about 350 nm, about 375 nm, about400 nm, about 425 nm, about 450 nm, about 475 nm, about 500 nm, about525 nm, about 550 nm, about 575 nm, about 600 nm, about 625 nm, about650 nm, about 700 nm, about 725 nm, about 750 nm, about 775 nm, about800 nm, about 825 nm, about 850 nm, about 875 nm, about 900 nm, about925 nm, about 950 nm, about 975 nm, or about 1000 nm. In an embodiment,the RNA lipoplex particles have an average diameter that ranges fromabout 250 nm to about 700 nm. In another embodiment, the RNA lipoplexparticles have an average diameter that ranges from about 300 nm toabout 500 nm. In an exemplary embodiment, the RNA lipoplex particleshave an average diameter of about 400 nm.

The RNA lipoplex particles and compositions comprising RNA lipoplexparticles described herein are useful for delivery of RNA to a targettissue after parenteral administration, in particular after intravenousadministration. The RNA lipoplex particles may be prepared usingliposomes that may be obtained by injecting a solution of the lipids inethanol into water or a suitable aqueous phase. In one embodiment, theaqueous phase has an acidic pH. In one embodiment, the aqueous phasecomprises acetic acid, e.g., in an amount of about 5 mM. Liposomes maybe used for preparing RNA lipoplex particles by mixing the liposomeswith RNA. In one embodiment, the liposomes and RNA lipoplex particlescomprise at least one cationic lipid and at least one additional lipid.In one embodiment, the at least one cationic lipid comprises1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and/or1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). In one embodiment, theat least one additional lipid comprises1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE),cholesterol (Chol) and/or 1,2-dioleoyl-sn-glycero-3-phosphocholine(DOPC). In one embodiment, the at least one cationic lipid comprises1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and the atleast one additional lipid comprises1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE). In oneembodiment, the liposomes and RNA lipoplex particles comprise1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE). Spleentargeting RNA lipoplex particles are described in WO 2013/143683, hereinincorporated by reference. It has been found that RNA lipoplex particleshaving a net negative charge may be used to preferentially target spleentissue or spleen cells such as antigen-presenting cells, in particulardendritic cells. Accordingly, following administration of the RNAlipoplex particles, RNA accumulation and/or RNA expression in the spleenoccurs. Thus, RNA lipoplex particles of the present disclosure may beused for expressing RNA in the spleen. In an embodiment, afteradministration of the RNA lipoplex particles, no or essentially no RNAaccumulation and/or RNA expression in the lung and/or liver occurs. Inone embodiment, after administration of the RNA lipoplex particles, RNAaccumulation and/or RNA expression in antigen presenting cells, such asprofessional antigen presenting cells in the spleen occurs. Thus, RNAlipoplex particles of the present disclosure may be used for expressingRNA in such antigen presenting cells. In one embodiment, the antigenpresenting cells are dendritic cells and/or macrophages.

Lipid Nanoparticles (LNPs)

In one embodiment, nucleic acid such as RNA described herein isadministered in the form of lipid nanoparticles (LNPs). The LNP maycomprise any lipid capable of forming a particle to which the one ormore nucleic acid molecules are attached, or in which the one or morenucleic acid molecules are encapsulated.

In one embodiment, the LNP comprises one or more cationic lipids, andone or more stabilizing lipids. Stabilizing lipids include neutrallipids and pegylated lipids.

In one embodiment, the LNP comprises a cationic lipid, a neutral lipid,a steroid, a polymer conjugated lipid; and the RNA, encapsulated withinor associated with the lipid nanoparticle. In one embodiment, the LNPcomprises from 40 to 55 mol percent, from 40 to 50 mol percent, from 41to 49 mol percent, from 41 to 48 mol percent, from 42 to 48 mol percent,from 43 to 48 mol percent, from 44 to 48 mol percent, from 45 to 48 molpercent, from 46 to 48 mol percent, from 47 to 48 mol percent, or from47.2 to 47.8 mol percent of the cationic lipid. In one embodiment, theLNP comprises about 47.0, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7,47.8, 47.9 or 48.0 mol percent of the cationic lipid.

In one embodiment, the neutral lipid is present in a concentrationranging from 5 to 15 mol percent, from 7 to 13 mol percent, or from 9 to11 mol percent. In one embodiment, the neutral lipid is present in aconcentration of about 9.5, 10 or 10.5 mol percent.

In one embodiment, the steroid is present in a concentration rangingfrom 30 to 50 mol percent, from 35 to 45 mol percent or from 38 to 43mol percent. In one embodiment, the steroid is present in aconcentration of about 40, 41, 42, 43, 44, 45 or 46 mol percent.

In one embodiment, the LNP comprises from 1 to 10 mol percent, from 1 to5 mol percent, or from 1 to 2.5 mol percent of the polymer conjugatedlipid.

In one embodiment, the LNP comprises from 40 to 50 mol percent acationic lipid; from 5 to mol percent of a neutral lipid; from 35 to 45mol percent of a steroid; from 1 to 10 mol percent of a polymerconjugated lipid; and the RNA, encapsulated within or associated withthe lipid nanoparticle.

In one embodiment, the mol percent is determined based on total mol oflipid present in the lipid nanoparticle.

In one embodiment, the neutral lipid is selected from the groupconsisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, DOPG, DPPG, POPE,DPPE, DMPE, DSPE, and SM. In one embodiment, the neutral lipid isselected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPEand SM. In one embodiment, the neutral lipid is DSPC.

In one embodiment, the steroid is cholesterol.

In one embodiment, the polymer conjugated lipid is a pegylated lipid. Inone embodiment, the pegylated lipid has the following structure:

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof,wherein:R¹² and R¹³ are each independently a straight or branched, saturated orunsaturated alkyl chain containing from 10 to 30 carbon atoms, whereinthe alkyl chain is optionally interrupted by one or more ester bonds;and w has a mean value ranging from 30 to 60. In one embodiment, R¹² andR¹³ are each independently straight, saturated alkyl chains containingfrom 12 to 16 carbon atoms. In one embodiment, w has a mean valueranging from 40 to 55. In one embodiment, the average w is about 45. Inone embodiment, R¹² and R¹³ are each independently a straight, saturatedalkyl chain containing about 14 carbon atoms, and w has a mean value ofabout 45.

In one embodiment, the pegylated lipid is DMG-PEG 2000, e.g., having thefollowing structure:

In some embodiments, the cationic lipid component of the LNPs has thestructure of Formula (III):

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomerthereof, wherein: one of L¹ or L² is —O(C═O)—, —(C═O)O—, —C(═O)—, —O—,—S(O)_(x)—, —S—S—, —C(═O)S—, SC(═O)—, —NR^(a)C(═O)—, —C(═O)NR^(a)—,NR^(a)C(═O)NR^(a)—, —OC(═O)NR^(a)— or —NR^(a)C(═O)O—, and the other ofL¹ or L² is —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O)_(x), —S—S—,—C(═O)S—, SC(═O)—, —NR^(a)C(═O)—, —C(═O)NR^(a)—, NR^(a)C(═O)NR^(a).—OC(═O)NR^(a)— or —NR^(a)C(═O)O— or a direct bond;G¹ and G² are each independently unsubstituted C₁-C₁₂ alkylene or C₁-C₁₂alkenylene;G³ is C₁-C₂₄ alkylene, C₁-C₂₄ alkenylene, C₃-C₈ cycloalkylene, C₃-C₈cycloalkenylene;R^(a) is H or C₁-C₁₂ alkyl;R¹ and R² are each independently C₆-C₂₄ alkyl or C₆-C₂₄ alkenyl;R³ is H, OR⁵, CN, —C(═O)OR⁴, —OC(═O)R⁴ or —NR⁵C(═O)R⁴;R⁴ is C₁-C₁₂ alkyl;R⁵ is H or C₁-C₆ alkyl; andx is 0, 1 or 2.

In some of the foregoing embodiments of Formula (III), the lipid has oneof the following structures (IIIA) or (IIIB):

wherein:A is a 3 to 8-membered cycloalkyl or cycloalkylene ring;R⁶ is, at each occurrence, independently H, OH or C₁-C₂₄ alkyl;n is an integer ranging from 1 to 15.

In some of the foregoing embodiments of Formula (III), the lipid hasstructure (IIIA), and in other embodiments, the lipid has structure(IIIB).

In other embodiments of Formula (III), the lipid has one of thefollowing structures (IIIC) or (IIID):

wherein y and z are each independently integers ranging from 1 to 12.

In any of the foregoing embodiments of Formula (III), one of L¹ or L² is—O(C═O)—. For example, in some embodiments each of L¹ and L² are—O(C═O)—. In some different embodiments of any of the foregoing, L¹ andL² are each independently —(C═O)O— or —O(C═O)—. For example, in someembodiments each of L¹ and L² is —(C═O)O—.

In some different embodiments of Formula (III), the lipid has one of thefollowing structures (IIIE) or (IIIF):

In some of the foregoing embodiments of Formula (III), the lipid has oneof the following structures (IIIG), (IIIH), (IIII), or (IIIJ):

In some of the foregoing embodiments of Formula (III), n is an integerranging from 2 to 12, for example from 2 to 8 or from 2 to 4. Forexample, in some embodiments, n is 3, 4, 5 or 6. In some embodiments, nis 3. In some embodiments, n is 4. In some embodiments, n is 5. In someembodiments, n is 6.

In some other of the foregoing embodiments of Formula (III), y and z areeach independently an integer ranging from 2 to 10. For example, in someembodiments, y and z are each independently an integer ranging from 4 to9 or from 4 to 6.

In some of the foregoing embodiments of Formula (III), R⁶ is H. In otherof the foregoing embodiments, R⁶ is C₁-C₂₄ alkyl. In other embodiments,R⁶ is OH.

In some embodiments of Formula (III), G³ is unsubstituted. In otherembodiments, G3 is substituted. In various different embodiments, G³ islinear C₁-C₂₄ alkylene or linear C₁-C₂₄ alkenylene.

In some other foregoing embodiments of Formula (III), R¹ or R², or both,is C₆-C₂₄ alkenyl. For example, in some embodiments, R¹ and R² each,independently have the following structure:

wherein:R^(7a) and R^(7b) are, at each occurrence, independently H or C₁-C₁₂alkyl; and a is an integer from 2 to 12,wherein R^(7a), R^(7b) and a are each selected such that R¹ and R² eachindependently comprise from 6 to 20 carbon atoms. For example, in someembodiments a is an integer ranging from 5 to 9 or from 8 to 12.

In some of the foregoing embodiments of Formula (III), at least oneoccurrence of R^(7a) is H. For example, in some embodiments, R^(7a) is Hat each occurrence. In other different embodiments of the foregoing, atleast one occurrence of R^(7b) is C₁-C₈ alkyl. For example, in someembodiments, C₁-C₈ alkyl is methyl, ethyl, n-propyl, iso-propyl,n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.

In different embodiments of Formula (III), R¹ or R², or both, has one ofthe following structures:

In some of the foregoing embodiments of Formula (III), R³ is OH, CN,—C(═O)OR⁴, —OC(═O)R⁴ or —NHC(═O)R⁴. In some embodiments, R⁴ is methyl orethyl.

In various different embodiments, the cationic lipid of Formula (III)has one of the structures set forth in the table below.

TABLE 17 Representative Compounds of Formula (III). No. Structure III-1

III-2

III-3

III-4

III-5

III-6

III-7

III-8

III-9

III-10

III-11

III-12

III-13

III-14

III-15

III-16

III-17

III-18

III-19

III-20

III-21

III-22

III-23

III-24

III-25

III-26

III-27

III-28

III-29

III-30

III-31

III-32

III-33

III-34

III-35

III-36

In some embodiments, the LNP comprises a lipid of Formula (III), RNA, aneutral lipid, a steroid and a pegylated lipid. In some embodiments, thelipid of Formula (III) is compound III-3. In some embodiments, theneutral lipid is DSPC. In some embodiments, the steroid is cholesterol.In some embodiments, the pegylated lipid is ALC-0159.

In some embodiments, the cationic lipid is present in the LNP in anamount from about 40 to about 50 mole percent. In one embodiment, theneutral lipid is present in the LNP in an amount from about 5 to about15 mole percent. In one embodiment, the steroid is present in the LNP inan amount from about 35 to about 45 mole percent. In one embodiment, thepegylated lipid is present in the LNP in an amount from about 1 to about10 mole percent.

In some embodiments, the LNP comprises compound III-3 in an amount fromabout 40 to about 50 mole percent, DSPC in an amount from about 5 toabout 15 mole percent, cholesterol in an amount from about 35 to about45 mole percent, and ALC-0159 in an amount from about 1 to about 10 molepercent.

In some embodiments, the LNP comprises compound III-3 in an amount ofabout 47.5 mole percent, DSPC in an amount of about 10 mole percent,cholesterol in an amount of about 40.7 mole percent, and ALC-0159 in anamount of about 1.8 mole percent.

In various different embodiments, the cationic lipid has one of thestructures set forth in the table below.

TABLE 18 Representative cationic lipids. No. Structure A

B

C

D

E

F

In some embodiments, the LNP comprises a cationic lipid shown in theabove table, e.g., a cationic lipid of Formula (B) or Formula (D), inparticular a cationic lipid of Formula (D), RNA, a neutral lipid, asteroid and a pegylated lipid. In some embodiments, the neutral lipid isDSPC. In some embodiments, the steroid is cholesterol. In someembodiments, the pegylated lipid is DMG-PEG 2000.

In one embodiment, the LNP comprises a cationic lipid that is anionizable lipid-like material (lipidoid). In one embodiment, thecationic lipid has the following structure:

The N/P value is preferably at least about 4. In some embodiments, theN/P value ranges from 4 to 20, 4 to 12, 4 to 10, 4 to 8, or 5 to 7. Inone embodiment, the N/P value is about 6.

LNP described herein may have an average diameter that in one embodimentranges from about 30 nm to about 200 nm, or from about 60 nm to about120 nm.

RNA Targeting

Some aspects of the present disclosure involve the targeted delivery ofthe RNA disclosed herein (e.g., RNA encoding vaccine antigens and/orimmunostimulants).

In one embodiment, the present disclosure involves targeting lung.Targeting lung is in particular preferred if the RNA administered is RNAencoding vaccine antigen. RNA may be delivered to lung, for example, byadministering the RNA which may be formulated as particles as describedherein, e.g., lipid particles, by inhalation.

In one embodiment, the present disclosure involves targeting thelymphatic system, in particular secondary lymphoid organs, morespecifically spleen. Targeting the lymphatic system, in particularsecondary lymphoid organs, more specifically spleen is in particularpreferred if the RNA administered is RNA encoding vaccine antigen.

In one embodiment, the target cell is a spleen cell. In one embodiment,the target cell is an antigen presenting cell such as a professionalantigen presenting cell in the spleen. In one embodiment, the targetcell is a dendritic cell in the spleen.

The “lymphatic system” is part of the circulatory system and animportant part of the immune system, comprising a network of lymphaticvessels that carry lymph. The lymphatic system consists of lymphaticorgans, a conducting network of lymphatic vessels, and the circulatinglymph. The primary or central lymphoid organs generate lymphocytes fromimmature progenitor cells. The thymus and the bone marrow constitute theprimary lymphoid organs. Secondary or peripheral lymphoid organs, whichinclude lymph nodes and the spleen, maintain mature naïve lymphocytesand initiate an adaptive immune response.

RNA may be delivered to spleen by so-called lipoplex formulations, inwhich the RNA is bound to liposomes comprising a cationic lipid andoptionally an additional or helper lipid to form injectable nanoparticleformulations. The liposomes may be obtained by injecting a solution ofthe lipids in ethanol into water or a suitable aqueous phase. RNAlipoplex particles may be prepared by mixing the liposomes with RNA.Spleen targeting RNA lipoplex particles are described in WO 2013/143683,herein incorporated by reference. It has been found that RNA lipoplexparticles having a net negative charge may be used to preferentiallytarget spleen tissue or spleen cells such as antigen-presenting cells,in particular dendritic cells. Accordingly, following administration ofthe RNA lipoplex particles, RNA accumulation and/or RNA expression inthe spleen occurs. Thus, RNA lipoplex particles of the presentdisclosure may be used for expressing RNA in the spleen. In anembodiment, after administration of the RNA lipoplex particles, no oressentially no RNA accumulation and/or RNA expression in the lung and/orliver occurs. In one embodiment, after administration of the RNAlipoplex particles, RNA accumulation and/or RNA expression in antigenpresenting cells, such as professional antigen presenting cells in thespleen occurs. Thus, RNA lipoplex particles of the present disclosuremay be used for expressing RNA in such antigen presenting cells. In oneembodiment, the antigen presenting cells are dendritic cells and/ormacrophages.

The electric charge of the RNA lipoplex particles of the presentdisclosure is the sum of the electric charges present in the at leastone cationic lipid and the electric charges present in the RNA. Thecharge ratio is the ratio of the positive charges present in the atleast one cationic lipid to the negative charges present in the RNA. Thecharge ratio of the positive charges present in the at least onecationic lipid to the negative charges present in the RNA is calculatedby the following equation: charge ratio=[(cationic lipid concentration(mol))*(the total number of positive charges in the cationiclipid)]/[(RNA concentration (mol))*(the total number of negative chargesin RNA)].

The spleen targeting RNA lipoplex particles described herein atphysiological pH preferably have a net negative charge such as a chargeratio of positive charges to negative charges from about 1.9:2 to about1:2, or about 1.6:2 to about 1:2, or about 1.6:2 to about 1.1:2. Inspecific embodiments, the charge ratio of positive charges to negativecharges in the RNA lipoplex particles at physiological pH is about1.9:2.0, about 1.8:2.0, about 1.7:2.0, about 1.6:2.0, about 1.5:2.0,about 1.4:2.0, about 1.3:2.0, about 1.2:2.0, about 1.1:2.0, or about1:2.0.

Immunostimulants may be provided to a subject by administering to thesubject RNA encoding an immunostimulant in a formulation forpreferential delivery of RNA to liver or liver tissue. The delivery ofRNA to such target organ or tissue is preferred, in particular, if it isdesired to express large amounts of the immunostimulant and/or ifsystemic presence of the immunostimulant, in particular in significantamounts, is desired or required.

RNA delivery systems have an inherent preference to the liver. Thispertains to lipid-based particles, cationic and neutral nanoparticles,in particular lipid nanoparticles such as liposomes, nanomicelles andlipophilic ligands in bioconjugates. Liver accumulation is caused by thediscontinuous nature of the hepatic vasculature or the lipid metabolism(liposomes and lipid or cholesterol conjugates).

For in vivo delivery of RNA to the liver, a drug delivery system may beused to transport the RNA into the liver by preventing its degradation.For example, polyplex nanomicelles consisting of a poly(ethylene glycol)(PEG)-coated surface and an RNA (e.g., mRNA)-containing core is a usefulsystem because the nanomicelles provide excellent in vivo stability ofthe RNA, under physiological conditions. Furthermore, the stealthproperty provided by the polyplex nanomicelle surface, composed of densePEG palisades, effectively evades host immune defenses.

Examples of suitable immunostimulants for targeting liver are cytokinesinvolved in T cell proliferation and/or maintenance. Examples ofsuitable cytokines include IL2 or IL7, fragments and variants thereof,and fusion proteins of these cytokines, fragments and variants, such asextended-PK cytokines.

In another embodiment, RNA encoding an immunostimulant may beadministered in a formulation for preferential delivery of RNA to thelymphatic system, in particular secondary lymphoid organs, morespecifically spleen. The delivery of an immunostimulant to such targettissue is preferred, in particular, if presence of the immunostimulantin this organ or tissue is desired (e.g., for inducing an immuneresponse, in particular in case immunostimulants such as cytokines arerequired during T-cell priming or for activation of resident immunecells), while it is not desired that the immunostimulant is presentsystemically, in particular in significant amounts (e.g., because theimmunostimulant has systemic toxicity).

Examples of suitable immunostimulants are cytokines involved in T cellpriming. Examples of suitable cytokines include IL12, IL15, IFN-α, orIFN-β, fragments and variants thereof, and fusion proteins of thesecytokines, fragments and variants, such as extended-PK cytokines.

Immunostimulants

In one embodiment, the RNA encoding vaccine antigen may benon-immunogenic. In this and other embodiments, the RNA encoding vaccineantigen may be co-administered with an immunostimulant or RNA encodingan immunostimulant. The methods and agents described herein areparticularly effective if the immunostimulant is attached to apharmacokinetic modifying group (hereafter referred to as“extended-pharmacokinetic (PK)” immunostimulant). The methods and agentsdescribed herein are particularly effective if the immunostimulant isadministered in the form of RNA encoding an immunostimulant. In oneembodiment, said RNA is targeted to the liver for systemic availability.Liver cells can be efficiently transfected and are able to produce largeamounts of protein.

An “immunostimulant” is any substance that stimulates the immune systemby inducing activation or increasing activity of any of the immunesystem's components, in particular immune effector cells. Theimmunostimulant may be pro-inflammatory.

According to one aspect, the immunostimulant is a cytokine or a variantthereof. Examples of cytokines include interferons, such asinterferon-alpha (IFN-α) or interferon-gamma (IFN-γ), interleukins, suchas IL2, IL7, IL12, IL15 and IL23, colony stimulating factors, such asM-CSF and GM-CSF, and tumor necrosis factor. According to anotheraspect, the immunostimulant includes an adjuvant-type immunostimulatoryagent such as APC Toll-like Receptor agonists or costimulatory/celladhesion membrane proteins. Examples of Toll-like Receptor agonistsinclude costimulatory/adhesion proteins such as CD80, CD86, and ICAM-1.

Cytokines are a category of small proteins (˜5-20 kDa) that areimportant in cell signaling. Their release has an effect on the behaviorof cells around them. Cytokines are involved in autocrine signaling,paracrine signaling and endocrine signaling as immunomodulating agents.Cytokines include chemokines, interferons, interleukins, lymphokines,and tumour necrosis factors but generally not hormones or growth factors(despite some overlap in the terminology). Cytokines are produced by abroad range of cells, including immune cells like macrophages, Blymphocytes, T lymphocytes and mast cells, as well as endothelial cells,fibroblasts, and various stromal cells. A given cytokine may be producedby more than one type of cell. Cytokines act through receptors, and areespecially important in the immune system; cytokines modulate thebalance between humoral and cell-based immune responses, and theyregulate the maturation, growth, and responsiveness of particular cellpopulations. Some cytokines enhance or inhibit the action of othercytokines in complex ways.

According to the present disclosure, a cytokine may be a naturallyoccurring cytokine or a functional fragment or variant thereof. Acytokine may be human cytokine and may be derived from any vertebrate,especially any mammal. One particularly preferred cytokine isinterferon-α.

Interferons

Interferons (IFNs) are a group of signaling proteins made and releasedby host cells in response to the presence of several pathogens, such asviruses, bacteria, parasites, and also tumor cells. In a typicalscenario, a virus-infected cell will release interferons causing nearbycells to heighten their anti-viral defenses.

Based on the type of receptor through which they signal, interferons aretypically divided among three classes: type I interferon, type IIinterferon, and type Ill interferon.

All type I interferons bind to a specific cell surface receptor complexknown as the IFN-α/β receptor (IFNAR) that consists of IFNAR1 and IFNAR2chains.

The type I interferons present in humans are IFNα, IFNβ, IFNε, IFNκ andIFNω. In general, type I interferons are produced when the bodyrecognizes a virus that has invaded it. They are produced by fibroblastsand monocytes. Once released, type I interferons bind to specificreceptors on target cells, which leads to expression of proteins thatwill prevent the virus from producing and replicating its RNA and DNA.

The IFNα proteins are produced mainly by plasmacytoid dendritic cells(pDCs). They are mainly involved in innate immunity against viralinfection. The genes responsible for their synthesis come in 13 subtypesthat are called IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10,IFNA13, IFNA14, IFNA16, IFNA17, IFNA21. These genes are found togetherin a cluster on chromosome 9.

The IFNβ proteins are produced in large quantities by fibroblasts. Theyhave antiviral activity that is involved mainly in innate immuneresponse. Two types of IFNβ have been described, IFNβ1 and IFNβ3. Thenatural and recombinant forms of IFNβ1 have antiviral, antibacterial,and anticancer properties.

Type II interferon (IFNγ in humans) is also known as immune interferonand is activated by IL12. Furthermore, type II interferons are releasedby cytotoxic T cells and T helper cells.

Type III interferons signal through a receptor complex consisting ofIL10R2 (also called CRF2-4) and IFNLR1 (also called CRF2-12). Althoughdiscovered more recently than type I and type II IFNs, recentinformation demonstrates the importance of type III IFNs in some typesof virus or fungal infections.

In general, type I and II interferons are responsible for regulating andactivating the immune response.

According to the present disclosure, a type I interferon is preferablyIFNα or IFNβ, more preferably IFNα.

According to the present disclosure, an interferon may be a naturallyoccurring interferon or a functional fragment or variant thereof. Aninterferon may be human interferon and may be derived from anyvertebrate, especially any mammal.

Interleukins

Interleukins (ILs) are a group of cytokines (secreted proteins andsignal molecules) that can be divided into four major groups based ondistinguishing structural features. However, their amino acid sequencesimilarity is rather weak (typically 15-25% identity). The human genomeencodes more than 50 interleukins and related proteins.

According to the present disclosure, an interleukin may be a naturallyoccurring interleukin or a functional fragment or variant thereof. Aninterleukin may be human interleukin and may be derived from anyvertebrate, especially any mammal.

Extended-PK Group

Immunostimulant polypeptides described herein can be prepared as fusionor chimeric polypeptides that include an immunostimulant portion and aheterologous polypeptide (i.e., a polypeptide that is not animmunostimulant). The immunostimulant may be fused to an extended-PKgroup, which increases circulation half-life. Non-limiting examples ofextended-PK groups are described infra. It should be understood thatother PK groups that increase the circulation half-life ofimmunostimulants such as cytokines, or variants thereof, are alsoapplicable to the present disclosure. In certain embodiments, theextended-PK group is a serum albumin domain (e.g., mouse serum albumin,human serum albumin).

As used herein, the term “PK” is an acronym for “pharmacokinetic” andencompasses properties of a compound including, byway of example,absorption, distribution, metabolism, and elimination by a subject. Asused herein, an “extended-PK group” refers to a protein, peptide, ormoiety that increases the circulation half-life of a biologically activemolecule when fused to or administered together with the biologicallyactive molecule. Examples of an extended-PK group include serum albumin(e.g., HSA), Immunoglobulin Fc or Fc fragments and variants thereof,transferrin and variants thereof, and human serum albumin (HSA) binders(as disclosed in U.S. Publication Nos. 2005/0287153 and 2007/0003549).Other exemplary extended-PK groups are disclosed in Kontermann, ExpertOpin Biol Ther, 2016 July; 16(7):903-which is herein incorporated byreference in its entirety. As used herein, an “extended-PK”immunostimulant refers to an immunostimulant moiety in combination withan extended-PK group. In one embodiment, the extended-PK immunostimulantis a fusion protein in which an immunostimulant moiety is linked orfused to an extended-PK group.

In certain embodiments, the serum half-life of an extended-PKimmunostimulant is increased relative to the immunostimulant alone(i.e., the immunostimulant not fused to an extended-PK group). Incertain embodiments, the serum half-life of the extended-PKimmunostimulant is at least 20, 40, 60, 80, 100, 120, 150, 180, 200,400, 600, 800, or 1000% longer relative to the serum half-life of theimmunostimulant alone. In certain embodiments, the serum half-life ofthe extended-PK immunostimulant is at least 1.5-fold, 2-fold, 2.5-fold,3-fold, 3.5 fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold,10-fold, 12-fold, 13-fold, 15-fold, 17-fold, 20-fold, 22-fold, 25-fold,27-fold, 30-fold, 35-fold, 40-fold, or 50-fold greater than the serumhalf-life of the immunostimulant alone. In certain embodiments, theserum half-life of the extended-PK immunostimulant is at least 10 hours,15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 50 hours, 60hours, 70 hours, 80 hours, 90 hours, 100 hours, 110 hours, 120 hours,130 hours, 135 hours, 140 hours, 150 hours, 160 hours, or 200 hours.

As used herein, “half-life” refers to the time taken for the serum orplasma concentration of a compound such as a peptide or protein toreduce by 50%, in vivo, for example due to degradation and/or clearanceor sequestration by natural mechanisms. An extended-PK immunostimulantsuitable for use herein is stabilized in vivo and its half-lifeincreased by, e.g., fusion to serum albumin (e.g., HSA or MSA), whichresist degradation and/or clearance or sequestration. The half-life canbe determined in any manner known per se, such as by pharmacokineticanalysis. Suitable techniques will be clear to the person skilled in theart, and may for example generally involve the steps of suitablyadministering a suitable dose of the amino acid sequence or compound toa subject; collecting blood samples or other samples from said subjectat regular intervals; determining the level or concentration of theamino acid sequence or compound in said blood sample; and calculating,from (a plot of) the data thus obtained, the time until the level orconcentration of the amino acid sequence or compound has been reduced by50% compared to the initial level upon dosing. Further details areprovided in, e.g., standard handbooks, such as Kenneth, A. et al.,Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and inPeters et al., Pharmacokinetic Analysis: A Practical Approach (1996).Reference is also made to Gibaldi, M. et al., Pharmacokinetics, 2nd Rev.Edition, Marcel Dekker (1982).

In certain embodiments, the extended-PK group includes serum albumin, orfragments thereof or variants of the serum albumin or fragments thereof(all of which for the purpose of the present disclosure are comprised bythe term “albumin”). Polypeptides described herein may be fused toalbumin (or a fragment or variant thereof) to form albumin fusionproteins. Such albumin fusion proteins are described in U.S. PublicationNo. 20070048282.

As used herein, “albumin fusion protein” refers to a protein formed bythe fusion of at least one molecule of albumin (or a fragment or variantthereof) to at least one molecule of a protein such as a therapeuticprotein, in particular an immunostimulant. The albumin fusion proteinmay be generated by translation of a nucleic acid in which apolynucleotide encoding a therapeutic protein is joined in-frame with apolynucleotide encoding an albumin. The therapeutic protein and albumin,once part of the albumin fusion protein, may each be referred to as a“portion”, “region” or “moiety” of the albumin fusion protein (e.g., a“therapeutic protein portion” or an “albumin protein portion”). In ahighly preferred embodiment, an albumin fusion protein comprises atleast one molecule of a therapeutic protein (including, but not limitedto a mature form of the therapeutic protein) and at least one moleculeof albumin (including but not limited to a mature form of albumin). Inone embodiment, an albumin fusion protein is processed by a host cellsuch as a cell of the target organ for administered RNA, e.g. a livercell, and secreted into the circulation. Processing of the nascentalbumin fusion protein that occurs in the secretory pathways of the hostcell used for expression of the RNA may include, but is not limited tosignal peptide cleavage; formation of disulfide bonds; proper folding;addition and processing of carbohydrates (such as for example, N- andO-linked glycosylation); specific proteolytic cleavages; and/or assemblyinto multimeric proteins. An albumin fusion protein is preferablyencoded by RNA in a non-processed form which in particular has a signalpeptide at its N-terminus and following secretion by a cell ispreferably present in the processed form wherein in particular thesignal peptide has been cleaved off. In a most preferred embodiment, the“processed form of an albumin fusion protein” refers to an albuminfusion protein product which has undergone N-terminal signal peptidecleavage, herein also referred to as a “mature albumin fusion protein”.In preferred embodiments, albumin fusion proteins comprising atherapeutic protein have a higher plasma stability compared to theplasma stability of the same therapeutic protein when not fused toalbumin. Plasma stability typically refers to the time period betweenwhen the therapeutic protein is administered in vivo and carried intothe bloodstream and when the therapeutic protein is degraded and clearedfrom the bloodstream, into an organ, such as the kidney or liver, thatultimately clears the therapeutic protein from the body. Plasmastability is calculated in terms of the half-life of the therapeuticprotein in the bloodstream. The half-life of the therapeutic protein inthe bloodstream can be readily determined by common assays known in theart.

As used herein, “albumin” refers collectively to albumin protein oramino acid sequence, or an albumin fragment or variant, having one ormore functional activities (e.g., biological activities) of albumin. Inparticular, “albumin” refers to human albumin or fragments or variantsthereof especially the mature form of human albumin, or albumin fromother vertebrates or fragments thereof, or variants of these molecules.The albumin may be derived from any vertebrate, especially any mammal,for example human, cow, sheep, or pig. Non-mammalian albumins include,but are not limited to, hen and salmon. The albumin portion of thealbumin fusion protein may be from a different animal than thetherapeutic protein portion.

In certain embodiments, the albumin is human serum albumin (HSA), orfragments or variants thereof, such as those disclosed in U.S. Pat. No.5,876,969, WO 2011/124718, WO 2013/075066, and WO 2011/0514789.

The terms, human serum albumin (HSA) and human albumin (HA) are usedinterchangeably herein. The terms, “albumin and “serum albumin” arebroader, and encompass human serum albumin (and fragments and variantsthereof) as well as albumin from other species (and fragments andvariants thereof).

As used herein, a fragment of albumin sufficient to prolong thetherapeutic activity or plasma stability of the therapeutic proteinrefers to a fragment of albumin sufficient in length or structure tostabilize or prolong the therapeutic activity or plasma stability of theprotein so that the plasma stability of the therapeutic protein portionof the albumin fusion protein is prolonged or extended compared to theplasma stability in the non-fusion state.

The albumin portion of the albumin fusion proteins may comprise the fulllength of the albumin sequence, or may include one or more fragmentsthereof that are capable of stabilizing or prolonging the therapeuticactivity or plasma stability. Such fragments may be of 10 or more aminoacids in length or may include about 15, 20, 25, 30, 50, or morecontiguous amino acids from the albumin sequence or may include part orall of specific domains of albumin. For instance, one or more fragmentsof HSA spanning the first two immunoglobulin-like domains may be used.In a preferred embodiment, the HSA fragment is the mature form of HSA.

Generally speaking, an albumin fragment or variant will be at least 100amino acids long, preferably at least 150 amino acids long.

According to the present disclosure, albumin may be naturally occurringalbumin or a fragment or variant thereof. Albumin may be human albuminand may be derived from any vertebrate, especially any mammal.

Preferably, the albumin fusion protein comprises albumin as theN-terminal portion, and a therapeutic protein as the C-terminal portion.Alternatively, an albumin fusion protein comprising albumin as theC-terminal portion, and a therapeutic protein as the N-terminal portionmay also be used. In other embodiments, the albumin fusion protein has atherapeutic protein fused to both the N-terminus and the C-terminus ofalbumin. In a preferred embodiment, the therapeutic proteins fused atthe N- and C-termini are the same therapeutic proteins. In anotherpreferred embodiment, the therapeutic proteins fused at the N- andC-termini are different therapeutic proteins. In one embodiment, thedifferent therapeutic proteins are both cytokines.

In one embodiment, the therapeutic protein(s) is (are) joined to thealbumin through (a) peptide linker(s). A linker peptide between thefused portions may provide greater physical separation between themoieties and thus maximize the accessibility of the therapeutic proteinportion, for instance, for binding to its cognate receptor. The linkerpeptide may consist of amino acids such that it is flexible or morerigid. The linker sequence may be cleavable by a protease or chemically.

As used herein, the term “Fc region” refers to the portion of a nativeimmunoglobulin formed by the respective Fc domains (or Fc moieties) ofits two heavy chains. As used herein, the term “Fc domain” refers to aportion or fragment of a single immunoglobulin (Ig) heavy chain whereinthe Fc domain does not comprise an Fv domain. In certain embodiments, anFc domain begins in the hinge region just upstream of the papaincleavage site and ends at the C-terminus of the antibody. Accordingly, acomplete Fc domain comprises at least a hinge domain, a CH2 domain, anda CH3 domain. In certain embodiments, an Fc domain comprises at leastone of: a hinge (e.g., upper, middle, and/or lower hinge region) domain,a CH2 domain, a CH3 domain, a CH4 domain, or a variant, portion, orfragment thereof. In certain embodiments, an Fc domain comprises acomplete Fc domain (i.e., a hinge domain, a CH2 domain, and a CH3domain). In certain embodiments, an Fc domain comprises a hinge domain(or portion thereof) fused to a CH3 domain (or portion thereof). Incertain embodiments, an Fc domain comprises a CH2 domain (or portionthereof) fused to a CH3 domain (or portion thereof). In certainembodiments, an Fc domain consists of a CH3 domain or portion thereof.In certain embodiments, an Fc domain consists of a hinge domain (orportion thereof) and a CH3 domain (or portion thereof). In certainembodiments, an Fc domain consists of a CH2 domain (or portion thereof)and a CH3 domain. In certain embodiments, an Fc domain consists of ahinge domain (or portion thereof) and a CH2 domain (or portion thereof).In certain embodiments, an Fc domain lacks at least a portion of a CH2domain (e.g., all or part of a CH2 domain). An Fc domain hereingenerally refers to a polypeptide comprising all or part of the Fcdomain of an immunoglobulin heavy-chain. This includes, but is notlimited to, polypeptides comprising the entire CH1, hinge, CH2, and/orCH3 domains as well as fragments of such peptides comprising only, e.g.,the hinge, CH2, and CH3 domain. The Fc domain may be derived from animmunoglobulin of any species and/or any subtype, including, but notlimited to, a human IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgMantibody. The Fc domain encompasses native Fc and Fc variant molecules.As set forth herein, it will be understood by one of ordinary skill inthe art that any Fc domain may be modified such that it varies in aminoacid sequence from the native Fc domain of a naturally occurringimmunoglobulin molecule. In certain embodiments, the Fc domain hasreduced effector function (e.g., FcγR binding).

The Fc domains of a polypeptide described herein may be derived fromdifferent immunoglobulin molecules. For example, an Fc domain of apolypeptide may comprise a CH2 and/or CH3 domain derived from an IgG1molecule and a hinge region derived from an IgG3 molecule. In anotherexample, an Fc domain can comprise a chimeric hinge region derived, inpart, from an IgG1 molecule and, in part, from an IgG3 molecule. Inanother example, an Fc domain can comprise a chimeric hinge derived, inpart, from an IgG1 molecule and, in part, from an IgG4 molecule.

In certain embodiments, an extended-PK group includes an Fc domain orfragments thereof or variants of the Fc domain or fragments thereof (allof which for the purpose of the present disclosure are comprised by theterm “Fc domain”). The Fc domain does not contain a variable region thatbinds to antigen. Fc domains suitable for use in the present disclosuremay be obtained from a number of different sources. In certainembodiments, an Fc domain is derived from a human immunoglobulin. Incertain embodiments, the Fc domain is from a human IgG1 constant region.It is understood, however, that the Fc domain may be derived from animmunoglobulin of another mammalian species, including for example, arodent (e.g. a mouse, rat, rabbit, guinea pig) or non-human primate(e.g. chimpanzee, macaque) species. Moreover, the Fc domain (or afragment or variant thereof) may be derived from any immunoglobulinclass, including IgM, IgG, IgD, IgA, and IgE, and any immunoglobulinisotype, including IgG1, IgG2, IgG3, and IgG4.

A variety of Fc domain gene sequences (e.g., mouse and human constantregion gene sequences) are available in the form of publicly accessibledeposits. Constant region domains comprising an Fc domain sequence canbe selected lacking a particular effector function and/or with aparticular modification to reduce immunogenicity. Many sequences ofantibodies and antibody-encoding genes have been published and suitableFc domain sequences (e.g. hinge, CH2, and/or CH3 sequences, or fragmentsor variants thereof) can be derived from these sequences using artrecognized techniques.

In certain embodiments, the extended-PK group is a serum albumin bindingprotein such as those described in US2005/0287153, US2007/0003549,US2007/0178082, US2007/0269422, US2010/0113339, WO2009/083804, andWO2009/133208, which are herein incorporated by reference in theirentirety. In certain embodiments, the extended-PK group is transferrin,as disclosed in U.S. Pat. Nos. 7,176,278 and 8,158,579, which are hereinincorporated by reference in their entirety. In certain embodiments, theextended-PK group is a serum immunoglobulin binding protein such asthose disclosed in US2007/0178082, US2014/0220017, and US2017/0145062,which are herein incorporated by reference in their entirety. In certainembodiments, the extended-PK group is a fibronectin (Fn)-based scaffolddomain protein that binds to serum albumin, such as those disclosed inUS2012/0094909, which is herein incorporated by reference in itsentirety. Methods of making fibronectin-based scaffold domain proteinsare also disclosed in US2012/0094909. A non-limiting example of aFn3-based extended-PK group is Fn3(HSA), i.e., a Fn3 protein that bindsto human serum albumin.

In certain aspects, the extended-PK immunostimulant, suitable for useaccording to the present disclosure, can employ one or more peptidelinkers. As used herein, the term “peptide linker” refers to a peptideor polypeptide sequence which connects two or more domains (e.g., theextended-PK moiety and an immunostimulant moiety) in a linear amino acidsequence of a polypeptide chain. For example, peptide linkers may beused to connect an immunostimulant moiety to a HSA domain.

Linkers suitable for fusing the extended-PK group to e.g. animmunostimulant are well known in the art. Exemplary linkers includeglycine-serine-polypeptide linkers, glycine-proline-polypeptide linkers,and proline-alanine polypeptide linkers. In certain embodiments, thelinker is a glycine-serine-polypeptide linker, i.e., a peptide thatconsists of glycine and serine residues.

In addition to, or in place of, the heterologous polypeptides describedabove, an immunostimulant polypeptide described herein can containsequences encoding a “marker” or “reporter”. Examples of marker orreporter genes include β-lactamase, chloramphenicol acetyltransferase(CAT), adenosine deaminase (ADA), aminoglycoside phosphotransferase,dihydrofolate reductase (DHFR), hygromycin-B-hosphotransferase (HPH),thymidine kinase (TK), β-galactosidase, and xanthine guaninephosphoribosyltransferase (XGPRT).

Pharmaceutical Compositions

The agents described herein may be administered in pharmaceuticalcompositions or medicaments and may be administered in the form of anysuitable pharmaceutical composition.

In one embodiment, the pharmaceutical composition described herein is animmunogenic composition for inducing an immune response againstcoronavirus in a subject. For example, in one embodiment, theimmunogenic composition is a vaccine.

In one embodiment of all aspects of the present disclosure, thecomponents described herein such as RNA encoding a vaccine antigen maybe administered in a pharmaceutical composition which may comprise apharmaceutically acceptable carrier and may optionally comprise one ormore adjuvants, stabilizers etc. In one embodiment, the pharmaceuticalcomposition is for therapeutic or prophylactic treatments, e.g., for usein treating or preventing a coronavirus infection.

The term “pharmaceutical composition” relates to a formulationcomprising a therapeutically effective agent, preferably together withpharmaceutically acceptable carriers, diluents and/or excipients. Saidpharmaceutical composition is useful for treating, preventing, orreducing the severity of a disease or disorder by administration of saidpharmaceutical composition to a subject. A pharmaceutical composition isalso known in the art as a pharmaceutical formulation.

The pharmaceutical compositions of the present disclosure may compriseone or more adjuvants or may be administered with one or more adjuvants.The term “adjuvant” relates to a compound which prolongs, enhances oraccelerates an immune response. Adjuvants comprise a heterogeneous groupof compounds such as oil emulsions (e.g., Freund's adjuvants), mineralcompounds (such as alum), bacterial products (such as Bordetellapertussis toxin), or immune-stimulating complexes. Examples of adjuvantsinclude, without limitation, LPS, GP96, CpG oligodeoxynucleotides,growth factors, and cytokines, such as monokines, lymphokines,interleukins, chemokines. The cytokines may be IL1, IL2, IL3, IL4, IL5,IL6, IL7, IL8, IL9, IL10, IL12, IFNα, IFNγ, GM-CSF, LT-a. Further knownadjuvants are aluminium hydroxide, Freund's adjuvant or oil such asMontanide® ISA51. Other suitable adjuvants for use in the presentdisclosure include lipopeptides, such as Pam3Cys.

The pharmaceutical compositions according to the present disclosure aregenerally applied in a “pharmaceutically effective amount” and in “apharmaceutically acceptable preparation”.

The term “pharmaceutically acceptable” refers to the non-toxicity of amaterial which does not interact with the action of the active componentof the pharmaceutical composition.

The term “pharmaceutically effective amount” or “therapeuticallyeffective amount” refers to the amount which achieves a desired reactionor a desired effect alone or together with further doses. In the case ofthe treatment of a particular disease, the desired reaction preferablyrelates to inhibition of the course of the disease. This comprisesslowing down the progress of the disease and, in particular,interrupting or reversing the progress of the disease. The desiredreaction in a treatment of a disease may also be delay of the onset or aprevention of the onset of said disease or said condition. An effectiveamount of the compositions described herein will depend on the conditionto be treated, the severeness of the disease, the individual parametersof the patient, including age, physiological condition, size and weight,the duration of treatment, the type of an accompanying therapy (ifpresent), the specific route of administration and similar factors.Accordingly, the doses administered of the compositions described hereinmay depend on various of such parameters. In the case that a reaction ina patient is insufficient with an initial dose, higher doses (oreffectively higher doses achieved by a different, more localized routeof administration) may be used.

The pharmaceutical compositions of the present disclosure may containsalts, buffers, preservatives, and optionally other therapeutic agents.In one embodiment, the pharmaceutical compositions of the presentdisclosure comprise one or more pharmaceutically acceptable carriers,diluents and/or excipients.

Suitable preservatives for use in the pharmaceutical compositions of thepresent disclosure include, without limitation, benzalkonium chloride,chlorobutanol, paraben and thimerosal. The term “excipient” as usedherein refers to a substance which may be present in a pharmaceuticalcomposition of the present disclosure but is not an active ingredient.Examples of excipients, include without limitation, carriers, binders,diluents, lubricants, thickeners, surface active agents, preservatives,stabilizers, emulsifiers, buffers, flavoring agents, or colorants.

The term “diluent” relates a diluting and/or thinning agent. Moreover,the term “diluent” includes any one or more of fluid, liquid or solidsuspension and/or mixing media. Examples of suitable diluents includeethanol, glycerol and water.

The term “carrier” refers to a component which may be natural,synthetic, organic, inorganic in which the active component is combinedin order to facilitate, enhance or enable administration of thepharmaceutical composition. A carrier as used herein may be one or morecompatible solid or liquid fillers, diluents or encapsulatingsubstances, which are suitable for administration to subject. Suitablecarrier include, without limitation, sterile water, Ringer, Ringerlactate, sterile sodium chloride solution, isotonic saline, polyalkyleneglycols, hydrogenated naphthalenes and, in particular, biocompatiblelactide polymers, lactide/glycolide copolymers orpolyoxyethylene/polyoxy-propylene copolymers. In one embodiment, thepharmaceutical composition of the present disclosure includes isotonicsaline.

Pharmaceutically acceptable carriers, excipients or diluents fortherapeutic use are well known in the pharmaceutical art, and aredescribed, for example, in Remington's Pharmaceutical Sciences, MackPublishing Co. (A. R Gennaro edit. 1985).

Pharmaceutical carriers, excipients or diluents can be selected withregard to the intended route of administration and standardpharmaceutical practice.

In one embodiment, pharmaceutical compositions described herein may beadministered intravenously, intraarterially, subcutaneously,intradermally or intramuscularly. In certain embodiments, thepharmaceutical composition is formulated for local administration orsystemic administration. Systemic administration may include enteraladministration, which involves absorption through the gastrointestinaltract, or parenteral administration. As used herein, “parenteraladministration” refers to the administration in any manner other thanthrough the gastrointestinal tract, such as by intravenous injection. Ina preferred embodiment, the pharmaceutical composition is formulated forintramuscular administration. In another embodiment, the pharmaceuticalcomposition is formulated for systemic administration, e.g., forintravenous administration.

The term “co-administering” as used herein means a process wherebydifferent compounds or compositions (e.g., RNA encoding an antigen andRNA encoding an immunostimulant) are administered to the same patient.The different compounds or compositions may be administeredsimultaneously, at essentially the same time, or sequentially.

The pharmaceutical compositions and products described herein may beprovided as a frozen concentrate for solution for injection, e.g., at aconcentration of 0.50 mg/mL. In one embodiment, for preparation ofsolution for injection, a drug product is thawed and diluted withisotonic sodium chloride solution (e.g., 0.9% NaCl, saline), e.g., by aone-step dilution process. In some embodiments, bacteriostatic sodiumchloride solution (e.g., 0.9% NaCl, saline) cannot be used as a diluent.In some embodiments, a diluted drug product is an off-white suspension.The concentration of the final solution for injection varies dependingon the respective dose level to be administered.

In one embodiment, administration is performed within 6 h after begin ofpreparation due to the risk of microbial contamination and consideringthe multiple-dose approach of the preparation process. In oneembodiment, in this period of 6 h, two conditions are allowed: roomtemperature for preparation, handling and transfer as well as 2 to 8° C.for storage. Compositions described herein may be shipped and/or storedunder temperature-controlled conditions, e.g., temperature conditions ofabout 4-5° C. or below, about −20° C. or below, −70° C.±10° C. (e.g.,−80° C. to −60° C.), e.g., utilizing a cooling system (e.g., that may beor include dry ice) to maintain the desired temperature. In oneembodiment, compositions described herein are shipped intemperature-controlled thermal shippers. Such shippers may contain aGPS-enabled thermal sensor to track the location and temperature of eachshipment. The compositions can be stored by refilling with, e.g., dryice.

Treatments

The present disclosure provides methods and agents for inducing anadaptive immune response against coronavirus in a subject comprisingadministering an effective amount of a composition comprising RNAencoding a coronavirus vaccine antigen described herein.

In one embodiment, the methods and agents described herein provideimmunity in a subject to coronavirus, coronavirus infection, or to adisease or disorder associated with coronavirus. The present disclosurethus provides methods and agents for treating or preventing theinfection, disease, or disorder associated with coronavirus.

In one embodiment, the methods and agents described herein areadministered to a subject having an infection, disease, or disorderassociated with coronavirus. In one embodiment, the methods and agentsdescribed herein are administered to a subject at risk for developingthe infection, disease, or disorder associated with coronavirus. Forexample, the methods and agents described herein may be administered toa subject who is at risk for being in contact with coronavirus. In oneembodiment, the methods and agents described herein are administered toa subject who lives in, traveled to, or is expected to travel to ageographic region in which coronavirus is prevalent. In one embodiment,the methods and agents described herein are administered to a subjectwho is in contact with or expected to be in contact with another personwho lives in, traveled to, or is expected to travel to a geographicregion in which coronavirus is prevalent. In one embodiment, the methodsand agents described herein are administered to a subject who hasknowingly been exposed to coronavirus through their occupation, or othercontact. In one embodiment, a coronavirus is SARS-CoV-2. In someembodiments, methods and agents described herein are administered to asubject with evidence of prior exposure to and/or infection withSARS-CoV-2 and/or an antigen or epitope thereof or cross-reactivetherewith. For example, in some embodiments, methods and agentsdescribed herein are administered to a subject in whom antibodies, Bcells, and/or T cells reactive with one or more epitopes of a SARS-CoV-2spike protein are detectable and/or have been detected.

For a composition to be useful as a vaccine, the composition must inducean immune response against the coronavirus antigen in a cell, tissue orsubject (e.g., a human). In some embodiments, the composition induces animmune response against the coronavirus antigen in a cell, tissue orsubject (e.g., a human). In some instances, the vaccine induces aprotective immune response in a mammal. The therapeutic compounds orcompositions of the present disclosure may be administeredprophylactically (i.e., to prevent a disease or disorder) ortherapeutically (i.e., to treat a disease or disorder) to subjectssuffering from, or at risk of (or susceptible to) developing a diseaseor disorder. Such subjects may be identified using standard clinicalmethods. In the context of the present disclosure, prophylacticadministration occurs prior to the manifestation of overt clinicalsymptoms of disease, such that a disease or disorder is prevented oralternatively delayed in its progression. In the context of the field ofmedicine, the term “prevent” encompasses any activity, which reduces theburden of mortality or morbidity from disease. Prevention can occur atprimary, secondary and tertiary prevention levels. While primaryprevention avoids the development of a disease, secondary and tertiarylevels of prevention encompass activities aimed at preventing theprogression of a disease and the emergence of symptoms as well asreducing the negative impact of an already established disease byrestoring function and reducing disease-related complications.

The term “dose” as used herein refers in general to a “dose amount”which relates to the amount of RNA administered per administration,i.e., per dosing.

In some embodiments, administration of an immunogenic composition orvaccine of the present disclosure may be performed by singleadministration or boosted by multiple administrations.

In some embodiments, a regimen described herein includes at least onedose. In some embodiments, a regimen includes a first dose and at leastone subsequent dose. In some embodiments, the first dose is the sameamount as at least one subsequent dose. In some embodiments, the firstdose is the same amount as all subsequent doses. In some embodiments,the first dose is a different amount as at least one subsequent dose. Insome embodiments, the first dose is a different amount than allsubsequent doses. In some embodiments, a regimen comprises two doses. Insome embodiments, a provided regimen consists of two doses. In someembodiments, a regimen comprises three doses.

In one embodiment, the present disclosure envisions administration of asingle dose. In one embodiment, the present disclosure envisionsadministration of a priming dose followed by one or more booster doses.The booster dose or the first booster dose may be administered 7 to 28days or 14 to 24 days following administration of the priming dose. Insome embodiments, a first booster dose may be administered 1 week to 3months (e.g., 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks) followingadministration of a priming dose. In some embodiments, a subsequentbooster dose may be administered at least 1 week or longer, including,e.g., at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, orlonger, following a preceding booster dose. In some embodiments,subsequent booster doses may be administered about 5-9 weeks or 6-8weeks apart. In some embodiments, at least one subsequent booster dose(e.g., after a first booster dose) may be administered at least 3 monthsor longer, including, e.g., at least 4 months, at least 5 months, atleast 6 months, at least 7 months, at least 8 months, at least 9 months,at least 10 months, or longer, following a preceding dose.

In some embodiments, a subsequent dose given to an individual (e.g., aspart of a primary regimen or booster regimen) can have the same amountof RNA as previously given to the individual. In some embodiments, asubsequent dose given to an individual (e.g., as part of a primaryregimen or booster regimen) can differ in the amount of RNA, as comparedto the amount previously given to the individual. For example, in someembodiments, a subsequent dose can be higher or lower than the priordose, for example, based on consideration of various factors, including,e.g., immunogenicity and/or reactogenicity induced by the prior dose,prevalence of the disease, etc. In some embodiments, a subsequent dosecan be higher than a prior dose by at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, or higher.In some embodiments, a subsequent dose can be higher than a prior doseby at least 1.5-fold, at least 2-fold, at least 2.5 fold, at least3-fold, or higher. In some embodiments, a subsequent dose can be higherthan a prior dose by at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80%, at least 90%, or higher. In someembodiments, a subsequent dose can be lower than a prior dose by atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70% or lower. In some embodiments, an amount the RNAdescribed herein from 0.1 μg to 300 μg, 0.5 μg to 200 μg, or 1 μg to 100μg, such as about 1 μg, about 2 μg, about 3 μg, about 10 μg, about 15μg, about 20 μg, about 25 μg, about 30 μg, about 35 μg, about 40 μg,about 45 μg, about 50 μg, about 55 μg, about 60 μg, about 70 μg, about80 μg, about 90 μg, or about 100 μg may be administered per dose (e.g.,in a given dose).

In some embodiments, an amount of the RNA described herein of 60 μg orlower, 55 μg or lower, 50 μg or lower, 45 μg or lower, 40 μg or lower,35 μg or lower, 30 μg or lower, 25 μg or lower, 20 μg or lower, 15 μg orlower, 10 μg or lower, 5 μg or lower, 3 μg or lower, 2.5 μg or lower, or1 μg or lower may be administered per dose (e.g., in a given dose).

In some embodiments, an amount of the RNA described herein of at least0.25 μg, at least 0.5 μg, at least 1 μg, at least 2 μg, at least 3 μg,at least 4 μg, at least 5 μg, at least 10 μg, at least 15 μg, at least20 μg, at least 25 μg, at least 30 μg, at least 40 μg, at least 50 μg,or at least 60 μg may be administered per dose (e.g., in a given dose).In some embodiments, an amount of the RNA described herein of at least 3ug may be administered in at least one of given doses. In someembodiments, an amount of the RNA described herein of at least 10 ug maybe administered in at least one of given doses. In some embodiments, anamount of the RNA described herein of at least 15 ug may be administeredin at least one of given doses. In some embodiments, an amount of theRNA described herein of at least 20 ug may be administered in at leastone of given doses. In some embodiments, an amount of the RNA describedherein of at least 25 ug may be administered in at least one of givendoses. In some embodiments, an amount of the RNA described herein of atleast 30 ug may be administered in at least one of given doses. In someembodiments, an amount of the RNA described herein of at least 50 ug maybe administered in at least one of given doses. In some embodiments, anamount of the RNA described herein of at least 60 ug may be administeredin at least one of given doses.

In some embodiments, combinations of aforementioned amounts may beadministered in a regimen comprising two or more doses (e.g., a priordose and a subsequent dose can be of different amounts as describedherein). In some embodiments, combinations of aforementioned amounts maybe administered in a primary regimen and a booster regimen (e.g.,different doses can be given in a primary regimen and a boosterregimen).

In some embodiments, an amount of the RNA described herein of 0.25 μg to60 μg, 0.5 μg to 55 μg, 1 g to 50 μg, 5 μg to 40 μg, or 10 μg to 30 μgmay be administered per dose. In some embodiments, an amount of the RNAdescribed herein of 3 μg to 30 μg may be administered in at least one ofgiven doses. In some embodiments, an amount of the RNA described hereinof 3 μg to 20 μg may be administered in at least one of given doses. Insome embodiments, an amount of the RNA described herein of 3 μg to 15 μgmay be administered in at least one of given doses. In some embodiments,an amount of the RNA described herein of 3 μg to 10 μg may beadministered in at least one of given doses. In some embodiments, anamount of the RNA described herein of 10 μg to 30 μg may be administeredin at least one of given doses.

In some embodiments, a regimen administered to a subject may comprise aplurality of doses (e.g., at least two doses, at least three doses, ormore). In some embodiments, a regimen administered to a subject maycomprise a first dose and a second dose, which are given at least 2weeks apart, at least 3 weeks apart, at least 4 weeks apart, or more. Insome embodiments, such doses may be at least 1 month, at least 2 months,at least 3 months, at least 4 months, at least 5 months, at least 6months, at least 7 months, at least 8 months, at least 9 months, atleast 10 months, at least 11 months, at least 12 months, or more apart.In some embodiments, doses may be administered days apart, such as 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60 or more days apart. In some embodiments, doses may beadministered about 1 to about 3 weeks apart, or about 1 to about 4 weeksapart, or about 1 to about 5 weeks apart, or about 1 to about 6 weeksapart, or about 1 to more than 6 weeks apart. In some embodiments, dosesmay be separated by a period of about 7 to about 60 days, such as forexample about 14 to about 48 days, etc. In some embodiments, a minimumnumber of days between doses may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or more. In some embodiments,a maximum number of days between doses may be about 60, 59, 58, 57, 56,55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38,37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, orfewer. In some embodiments, doses may be about 21 to about 28 daysapart. In some embodiments, doses may be about 19 to about 42 daysapart. In some embodiments, doses may be about 7 to about 28 days apart.In some embodiments, doses may be about 14 to about 24 days. In someembodiments, doses may be about 21 to about 42 days.

In some embodiments, a vaccination regimen comprises a first dose and asecond dose. In some embodiments, a first dose and a second dose areadministered by at least 21 days apart. In some embodiments, a firstdose and a second dose are administered by at least 28 days apart.

In some embodiments, a vaccination regimen comprises a first dose and asecond dose, wherein the amount of RNA administered in the first dose isthe same as the amount of RNA administered in the second dose. In someembodiments, a vaccination regimen comprises a first dose and a seconddose wherein the amount of RNA administered in the first dose differsfrom that administered in the second dose.

In some embodiments, a vaccination regimen comprises a first dose and asecond dose, wherein the amount of RNA administered in the first dose isless than that administered in the second dose. In some embodiments, theamount of RNA administered in the first dose is 10%-90% of the seconddose. In some embodiments, the amount of RNA administered in the firstdose is 10%-50% of the second dose. In some embodiments, the amount ofRNA administered in the first dose is 10%-20% of the second dose. Insome embodiments, the first dose and the second dose are administered atleast 2 weeks apart, including, at least 3 weeks apart, at least 4 weeksapart, at least 5 weeks apart, at least 6 weeks apart or longer. In someembodiments, the first dose and the second dose are administered atleast 3 weeks apart.

In some embodiments, a first dose comprises less than about 30 ug of RNAand a second dose comprises at least about 30 ug of RNA. In someembodiments, a first dose comprises about 1 to less than about 30 ug ofRNA (e.g., about 0.1, about 1, about 3, about 5, about 10, about 15,about 20, about 25, or less than about 30 ug of RNA) and a second dosecomprises about 30 to about 100 ug of RNA (e.g., about 30, about 40,about 50, or about 60 ug of RNA). In some embodiments, a first dosecomprises about 1 to about 20 ug of RNA, about 1 to about 10 ug of RNA,or about 1 to about 5 ug of RNA and a second dose comprises about 30 toabout 60 ug of RNA.

In some embodiments, a first dose comprises about 1 to about 10 ug ofRNA (e.g., about 1, about 2, about 3, about 4, about 5, about 6, about7, about 8, about 9, or about 10 ug of RNA) and a second dose comprisesabout 30 to about 60 ug of RNA (e.g., about 30, about 35, about 40,about 45, about 50, about 55, or about 60 ug of RNA).

In some embodiments, a first dose comprises about 1 ug of RNA and asecond dose comprises about 30 ug of RNA. In some embodiments, a firstdose comprises about 3 ug of RNA and a second dose comprises about 30 ugof RNA. In some embodiments, a first dose comprises about 5 ug of RNAand a second dose comprises about 30 ug of RNA. In some embodiments, afirst dose comprises about 10 ug of RNA and a second dose comprisesabout 30 ug of RNA. In some embodiments, a first dose comprises about 15ug of RNA and a second dose comprises about 30 ug of RNA.

In some embodiments, a first dose comprises about 1 ug of RNA and asecond dose comprises about 60 ug of RNA. In some embodiments, a firstdose comprises about 3 ug of RNA and a second dose comprises about 60 ugof RNA. In some embodiments, a first dose comprises about 5 ug of RNAand a second dose comprises about 60 ug of RNA. In some embodiments, afirst dose comprises about 6 ug of RNA and a second dose comprises about60 ug of RNA. In some embodiments, a first dose comprises about 10 ug ofRNA and a second dose comprises about 60 ug of RNA. In some embodiments,a first dose comprises about 15 ug of RNA and a second dose comprisesabout 60 ug of RNA. In some embodiments, a first dose comprises about 20ug of RNA and a second dose comprises about 60 ug of RNA. In someembodiments, a first dose comprises about 25 ug of RNA and a second dosecomprises about 60 ug of RNA. In some embodiments, a first dosecomprises about 30 ug of RNA and a second dose comprises about 60 ug ofRNA.

In some embodiments, a first dose comprises less than about 10 ug of RNAand a second dose comprises at least about 10 ug of RNA. In someembodiments, a first dose comprises about 0.1 to less than about 10 ugof RNA (e.g., about 0.1, about 0.5, about 1, about 2, about 3, about 4,about 5, about 6, about 7, about 8, or less than about 10 ug of RNA) anda second dose comprises about 10 to about 30 ug of RNA (e.g., about 10,about 15, about 20, about 25, or about 30 ug of RNA). In someembodiments, a first dose comprises about 0.1 to about 10 ug of RNA,about 1 to about 5 ug of RNA, or about 0.1 to about 3 ug of RNA and asecond dose comprises about 10 to about 30 ug of RNA.

In some embodiments, a first dose comprises about 0.1 to about 5 ug ofRNA (e.g., about 0.1, about 0.5, about 1, about 2, about 3, about 4,about 5 ug of RNA) and a second dose comprises about 10 to about 20 ugof RNA (e.g., about 10, about 12, about 14, about 16, about 18, about 20ug of RNA).

In some embodiments, a first dose comprises about 0.1 ug of RNA and asecond dose comprises about 10 ug of RNA. In some embodiments, a firstdose comprises about 0.3 ug of RNA and a second dose comprises about 10ug of RNA. In some embodiments, a first dose comprises about 1 ug of RNAand a second dose comprises about 10 ug of RNA. In some embodiments, afirst dose comprises about 3 ug of RNA and a second dose comprises about10 ug of RNA.

In some embodiments, a first dose comprises less than about 3 ug of RNAand a second dose comprises at least about 3 ug of RNA. In someembodiments, a first dose comprises about 0.1 to less than about 3 ug ofRNA (e.g., about 0.1, about 0.2, about 0.3, about 0.5, about 0.6, about0.7, about 0.8, about 0.9, about 1.0, about 1.5, about 2.0, or about 2.5ug of RNA) and a second dose comprises about 3 to about 10 ug of RNA(e.g., about 3, about 4, about 5, about 6, or about 7, about 8, about 9,or about 10 ug of RNA). In some embodiments, a first dose comprisesabout 0.1 to about 3 ug of RNA, about 0.1 to about 1 ug of RNA, or about0.1 to about 0.5 ug of RNA and a second dose comprises about 3 to about10 ug of RNA.

In some embodiments, a first dose comprises about 0.1 to about 1.0 ug ofRNA (e.g., about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about0.6, about 0.7, about 0.8, about 0.9, or about 1.0 ug of RNA) and asecond dose comprises about 1 to about 3 ug of RNA (e.g., about 1.0,about 1.5, about 2.0, about 2.5, or about 3.0 ug of RNA).

In some embodiments, a first dose comprises about 0.1 ug of RNA and asecond dose comprises about 3 ug of RNA. In some embodiments, a firstdose comprises about 0.3 ug of RNA and a second dose comprises about 3ug of RNA. In some embodiments, a first dose comprises about 0.5 ug ofRNA and a second dose comprises about 3 ug of RNA. In some embodiments,a first dose comprises about 1 ug of RNA and a second dose comprisesabout 3 ug of RNA.

In some embodiments, a vaccination regimen comprises a first dose and asecond dose, wherein the amount of RNA administered in the first dose isgreater than that administered in the second dose. In some embodiments,the amount of RNA administered in the second dose is 10%-90% of thefirst dose. In some embodiments, the amount of RNA administered in thesecond dose is 10%-50% of the first dose. In some embodiments, theamount of RNA administered in the second dose is 10%-20% of the firstdose. In some embodiments, the first dose and the second dose areadministered at least 2 weeks apart, including, at least 3 weeks apart,at least 4 weeks apart, at least 5 weeks apart, at least 6 weeks apartor longer. In some embodiments, the first dose and the second dose areadministered at least 3 weeks apart

In some embodiments, a first dose comprises at least about 30 ug of RNAand a second dose comprises less than about 30 ug of RNA. In someembodiments, a first dose comprises about 30 to about 100 ug of RNA(e.g., about 30, about 40, about 50, or about 60 ug of RNA) and a seconddose comprises about 1 to about 30 ug of RNA (e.g., about 0.1, about 1,about 3, about 5, about 10, about 15, about 20, about 25, or about 30 ugof RNA). In some embodiments, a second dose comprises about 1 to about20 ug of RNA, about 1 to about 10 ug of RNA, or about 1 to 5 ug of RNA.In some embodiments, a first dose comprises about 30 to about 60 ug ofRNA and a second dose comprises about 1 to about 20 ug of RNA, about 1to about 10 ug of RNA, or about 0.1 to about 3 ug of RNA.

In some embodiments, a first dose comprises about 30 to about 60 ug ofRNA (e.g., about 30, about 35, about 40, about 45, about 50, about 55,or about 60 ug of RNA) and a second dose comprises about 1 to about 10ug of RNA (e.g., about 1, about 2, about 3, about 4, about 5, about 6,about 7, about 8, about 9, or about 10 ug of RNA).

In some embodiments, a first dose comprises about 30 ug of RNA and asecond dose comprises about 1 ug of RNA. In some embodiments, a firstdose comprises about 30 ug of RNA and a second dose comprises about 3 ugof RNA. In some embodiments, a first dose comprises about 30 ug of RNAand a second dose comprises about 5 ug of RNA. In some embodiments, afirst dose comprises about 30 ug of RNA and a second dose comprisesabout 10 ug of RNA. In some embodiments, a first dose comprises about 30ug of RNA and a second dose comprises about 15 ug of RNA.

In some embodiments, a first dose comprises about 60 ug of RNA and asecond dose comprises about 1 ug of RNA. In some embodiments, a firstdose comprises about 60 ug of RNA and a second dose comprises about 3 ugof RNA. In some embodiments, a first dose comprises about 60 ug of RNAand a second dose comprises about 5 ug of RNA. In some embodiments, afirst dose comprises about 60 ug of RNA and a second dose comprisesabout 6 ug of RNA. In some embodiments, a first dose comprises about 60ug of RNA and a second dose comprises about 10 ug of RNA. In someembodiments, a first dose comprises about 60 ug of RNA and a second dosecomprises about 15 ug of RNA. In some embodiments, a first dosecomprises about 60 ug of RNA and a second dose comprises about 20 ug ofRNA. In some embodiments, a first dose comprises about 60 ug of RNA anda second dose comprises about 25 ug of RNA. In some embodiments, a firstdose comprises about 60 ug of RNA and a second dose comprises about 30ug of RNA.

In some embodiments, a first dose comprises at least about 10 ug of RNAand a second dose comprises less than about 10 ug of RNA. In someembodiments, a first dose comprises about 10 to about 30 ug of RNA(e.g., about 10, about 15, about 20, about 25, or about 30 ug of RNA)and a second dose comprises about 0.1 to less than about 10 ug of RNA(e.g., about 0.1, about 0.5, about 1, about 2, about 3, about 4, about5, about 6, about 7, about 8, or less than about 10 ug of RNA). In someembodiments, a first dose comprises about 10 to about 30 ug of RNA, orabout 0.1 to about 3 ug of RNA and a second dose comprises about 1 toabout 10 ug of RNA, or about 1 to about 5 ug of RNA.

In some embodiments, a first dose comprises about 10 to about 20 ug ofRNA (e.g., about 10, about 12, about 14, about 16, about 18, about 20 ugof RNA) and a second dose comprises about 0.1 to about 5 ug of RNA(e.g., about 0.1, about 0.5, about 1, about 2, about 3, about 4, orabout 5 ug of RNA).

In some embodiments, a first dose comprises about 10 ug of RNA and asecond dose comprises about 0.1 ug of RNA. In some embodiments, a firstdose comprises about 10 ug of RNA and a second dose comprises about 0.3ug of RNA. In some embodiments, a first dose comprises about 10 ug ofRNA and a second dose comprises about 1 ug of RNA. In some embodiments,a first dose comprises about 10 ug of RNA and a second dose comprisesabout 3 ug of RNA. In some embodiments, a first dose comprises at leastabout 3 ug of RNA and a second dose comprises less than about 3 ug ofRNA. In some embodiments, a first dose comprises about 3 to about 10 ugof RNA (e.g., about 3, about 4, about 5, about 6, or about 7, about 8,about 9, or about 10 ug of RNA) and a second dose comprises 0.1 to lessthan about 3 ug of RNA (e.g., about 0.1, about 0.2, about 0.3, about0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.5about 2.0, or about 2.5 ug of RNA). In some embodiments, a first dosecomprises about 3 to about 10 ug of RNA and a second dose comprisesabout 0.1 to about 3 ug of RNA, about 0.1 to about 1 ug of RNA, or about0.1 to about 0.5 ug of RNA.

In some embodiments, a first dose comprises about 1 to about 3 ug of RNA(e.g., about 1, about 1.5, about 2.0, about 2.5, or about 3.0 ug of RNA)and a second dose comprises about 0.1 to 0.3 ug of RNA (e.g., about 0.1,about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about0.8, about 0.9, or about 1.0 ug of RNA).

In some embodiments, a first dose comprises about 3 ug of RNA and asecond dose comprises about 0.1 ug of RNA. In some embodiments, a firstdose comprises about 3 ug of RNA and a second dose comprises about 0.3ug of RNA. In some embodiments, a first dose comprises about 3 ug of RNAand a second dose comprises about 0.6 ug of RNA. In some embodiments, afirst dose comprises about 3 ug of RNA and a second dose comprises about1 ug of RNA.

In some embodiments, a vaccination regimen comprises at least two doses,including, e.g., at least three doses, at least four doses or more. Insome embodiments, a vaccination regimen comprises three doses. In someembodiments, the time interval between the first dose and the seconddose can be the same as the time interval between the second dose andthe third dose. In some embodiments, the time interval between the firstdose and the second dose can be longer than the time interval betweenthe second dose and the third dose, e.g., by days or weeks (including,e.g., at least 3 days, at least 4 days, at least 5 days, at least 6days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4weeks, at least 5 weeks, at least 6 weeks, or longer). In someembodiments, the time interval between the first dose and the seconddose can be shorter than the time interval between the second dose andthe third dose, e.g., by days or weeks (including, e.g., at least 3days, at least 4 days, at least 5 days, at least 6 days, at least 1week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5weeks, at least 6 weeks, or longer). In some embodiments, the timeinterval between the first dose and the second dose can be shorter thanthe time interval between the second dose and the third dose, e.g., byat least 1 month (including, e.g., at least 2 months, at least 3 months,at least 4 months, at least 5 months, at least 6 months, at least 7months, at least 8 months, at least 9 months, at least 10 months, atleast 11 months, at least 12 months, or longer).

In some embodiments, a last dose of a primary regimen and a first doseof a booster regimen are given at least 2 months, at least 3 months, atleast 4 months, at least 5 months, at least 6 months, at least 7 months,at least 8 months, at least 9 months, at least 10 months, at least 11months, at least 12 months, or more apart. In some embodiments, aprimary regimen may comprises two doses. In some embodiments, a primaryregimen may comprises three doses. In some embodiments, a first dose anda second dose (and/or other subsequent dose) may be administered byintramuscular injection. In some embodiments, a first dose and a seconddose (and/or other subsequent dose) may be administered in the deltoidmuscle. In some embodiments, a first dose and a second dose (and/orother subsequent dose) may be administered in the same arm.

In some embodiments, an RNA (e.g., mRNA) composition described herein isadministered (e.g., by intramuscular injection) as a series of two doses(e.g., 0.3 mL each) 21 days apart. In some embodiments, an RNA (e.g.,mRNA) composition described herein is administered (e.g., byintramuscular injection) as a series of two doses (e.g., 0.2 mL each) 21days apart. In some embodiments, an RNA (e.g., mRNA) compositiondescribed herein is administered (e.g., by intramuscular injection) as aseries of three doses (e.g., 0.3 ml or lower including, e.g., 0.2 mL),wherein doses are given at least 3 weeks apart. In some embodiments, thefirst and second doses may be administered 3 weeks apart, while thesecond and third doses may be administered at a longer time intervalthan that between the first and the second doses, e.g., at least 4 weeksapart or longer (including, at least 5 weeks, at least 6 weeks, at least7 weeks, at least 8 weeks, at least 9 weeks, or longer). In someembodiments, each dose is about 60 ug. In some embodiments, each dose isabout 50 ug. In some embodiments, each dose is about 30 ug. In someembodiments, each dose is about 25 ug. In some embodiments, each dose isabout 20 ug. In some embodiments, each dose is about 15 ug. In someembodiments, each dose is about 10 ug. In some embodiments, each dose isabout 3 ug.

In some embodiments, at least one dose given in a vaccination regimen(e.g., a primary vaccination regimen and/or a booster vaccinationregimen) is about 60 ug. In some embodiments, at least one dose given ina vaccination regimen (e.g., a primary vaccination regimen and/or abooster vaccination regimen) is about 50 ug. In some embodiments, atleast one dose given in a vaccination regimen (e.g., a primaryvaccination regimen and/or a booster vaccination regimen) is about 30ug. In some embodiments, at least one dose given in a vaccinationregimen (e.g., a primary vaccination regimen and/or a boostervaccination regimen) is about 25 ug. In some embodiments, at least onedose given in a vaccination regimen (e.g., a primary vaccination regimenand/or a booster vaccination regimen) is about 20 ug. In someembodiments, at least one dose given in a vaccination regimen (e.g., aprimary vaccination regimen and/or a booster vaccination regimen) isabout 15 ug. In some embodiments, at least one dose given in avaccination regimen (e.g., a primary vaccination regimen and/or abooster vaccination regimen) is about 10 ug. In some embodiments, atleast one dose given in a vaccination regimen (e.g., a primaryvaccination regimen and/or a booster vaccination regimen) is about 3 ug.

In one embodiment, an amount of the RNA described herein of about 60 μgis administered per dose. In one embodiment, an amount of the RNAdescribed herein of about 50 μg is administered per dose. In oneembodiment, an amount of the RNA described herein of about 30 μg isadministered per dose. In one embodiment, an amount of the RNA describedherein of about 25 μg is administered per dose. In one embodiment, anamount of the RNA described herein of about 20 μg is administered perdose. In one embodiment, an amount of the RNA described herein of about15 μg is administered per dose. In one embodiment, an amount of the RNAdescribed herein of about 10 μg is administered per dose. In oneembodiment, an amount of the RNA described herein of about 5 μg isadministered per dose. In one embodiment, an amount of the RNA describedherein of about 3 μg is administered per dose. In one embodiment, atleast two of such doses are administered. For example, a second dose maybe administered about 21 days following administration of the firstdose.

In some embodiments, the efficacy of the RNA vaccine described herein(e.g., administered in two doses, wherein a second dose may beadministered about 21 days following administration of the first dose,and administered, for example, in an amount of about 30 μg per dose) isat least 70%, at least 80%, at least 90, or at least 95% beginning 7days after administration of the second dose (e.g., beginning 28 daysafter administration of the first dose if a second dose is administered21 days following administration of the first dose). In someembodiments, such efficacy is observed in populations of age of at least50, at least 55, at least 60, at least 65, at least 70, or older. Insome embodiments, the efficacy of the RNA vaccine described herein(e.g., administered in two doses, wherein a second dose may beadministered about 21 days following administration of the first dose,and administered, for example, in an amount of about 30 μg per dose)beginning 7 days after administration of the second dose (e.g.,beginning 28 days after administration of the first dose if a seconddose is administered 21 days following administration of the first dose)in populations of age of at least 65, such as 65 to 80, 65 to 75, or 65to 70, is at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, or at least 95%. Such efficacy may be observed over timeperiods of up to 1 month, 2 months, 3 months, 6 months or even longer.

In one embodiment, vaccine efficacy is defined as the percent reductionin the number of subjects with evidence of infection (vaccinatedsubjects vs. non-vaccinated subjects).

In one embodiment, efficacy is assessed through surveillance forpotential cases of COVID-19. If, at any time, a patient develops acuterespiratory illness, for the purposes herein, the patient can beconsidered to potentially have COVID-19 illness. The assessments caninclude a nasal (midturbinate) swab, which may be tested using a reversetranscription-polymerase chain reaction (RT-PCR) test to detectSARS-CoV-2. In addition, clinical information and results from localstandard-of-care tests can be assessed.

In some embodiments, efficacy assessments may utilize a definition ofSARS-CoV-2-related cases wherein:

-   -   Confirmed COVID-19: presence of at least 1 of the following        symptoms and SARS-CoV-2 NAAT (nucleic acid amplification-based        test) positive during, or within 4 days before or after, the        symptomatic period: fever; new or increased cough; new or        increased shortness of breath; chills; new or increased muscle        pain; new loss of taste or smell; sore throat; diarrhea;        vomiting. Alternatively or additionally, in some embodiments,        efficacy assessments may utilize a definition of        SARS-CoV-2-related cases wherein one or more of the following        additional symptoms defined by the CDC can be considered:        fatigue; headache; nasal congestion or runny nose; nausea.

In some embodiments, efficacy assessments may utilize a definition ofSARS-CoV-2-related severe cases

-   -   Confirmed severe COVID-19: confirmed COVID-19 and presence of at        least 1 of the following: clinical signs at rest indicative of        severe systemic illness (e.g., RR 230 breaths per minute, HR        2125 beats per minute, SpO₂593% on room air at sea level, or        PaO₂/FiO₂<300 mm Hg); respiratory failure (which can be defined        as needing high-flow oxygen, noninvasive ventilation, mechanical        ventilation, or ECMO); evidence of shock (e.g., SBP <90 mm Hg,        DBP <60 mm Hg, or requiring vasopressors); significant acute        renal, hepatic, or neurologic dysfunction; admission to an ICU;        death.

Alternatively or additionally, in some embodiments a serologicaldefinition can be used for patients without clinical presentation ofCOVID-19: e.g., confirmed seroconversion to SARS-CoV-2 without confirmedCOVID-19: e.g., positive N-binding antibody result in a patient with aprior negative N-binding antibody result.

In some embodiments, any or all of the following assays can be performedon serum samples: SARS-CoV-2 neutralization assay; S1-binding IgG levelassay; RBD-binding IgG level assay; N-binding antibody assay.

In one embodiment, methods and agents described herein are administeredto a paediatric population. In various embodiments, the paediatricpopulation comprises or consists of subjects under 18 years, e.g., 5 toless than 18 years of age, 12 to less than 18 years of age, 16 to lessthan 18 years of age, 12 to less than 16 years of age, 5 to less than 12years of age, or 6 months to less than 12 years of age. In variousembodiments, the paediatric population comprises or consists of subjectsunder 5 years, e.g., 2 to less than 5 years of age, 12 to less than 24months of age, 7 to less than 12 months of age, or less than 6 months ofage. In some such embodiments, an RNA (e.g., mRNA) composition describedherein is administered to subjects of less than 2 years old, forexample, 6 months to less than 2 years old. In some such embodiments, anRNA (e.g., mRNA) composition described herein is administered tosubjects of less than 6 months old, for example, 1 month to less than 4months old. In some embodiments, a dosing regimen (e.g., doses and/ordosing schedule) for a paediatric population may vary for different agegroups. For example, in some embodiments, a subject 6 months through 4years of age may be administered according to a primary regimencomprising at least three doses, in which the initial two doses areadministered at least 3 weeks (including, e.g., at least 4 weeks, atleast 5 weeks, at least 6 weeks, or longer) apart followed by a thirddose administered at least 8 weeks (including, e.g., at least 9 weeks,at least 10 weeks, at least 11 weeks, at least 12 weeks, or longer)after the second dose. In some such embodiments, at least one doseadministered is 3 ug RNA described herein. In some embodiments, asubject 5 years of age and older may be administered according to aprimary regimen comprising at least two doses, in which the two dosesare administered at least 3 weeks (including, e.g., at least 3 weeks, atleast 4 weeks, at least 5 weeks, at least 6 weeks, or longer) apart. Insome such embodiments, at least one dose administered is 10 ug RNAdescribed herein. In some embodiments, a subject 5 years of age andolder who are immunocompromised (e.g., in some embodiments subjects whohave undergone solid organ transplantation, or who are diagnosed withconditions that are considered to have an equivalent ofimmunocompromise) may be administered according to a primary regimencomprising at least three doses, in which the initial two doses areadministered at least 3 weeks (including, e.g., at least 3 weeks, atleast 4 weeks, at least 5 weeks, at least 6 weeks, or longer) apart,followed by a third dose administered at least 4 weeks (including, e.g.,at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks,at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12weeks, or longer) after the second dose.

In some embodiments, an RNA (e.g., mRNA) composition described herein isadministered to subjects of age 12 or older and each dose is about 30ug. In some embodiments, an RNA (e.g., mRNA) composition describedherein is administered to subjects of age 12 or older (including, e.g.,age 18 or older) and each dose is higher than 30 ug, including, e.g., 35ug, 40 ug, 45 ug, 50 ug, 55 ug, 60 ug, 65 ug, 70 ug, or higher. In somesuch embodiments, an RNA (e.g., mRNA) composition described herein isadministered to subjects of age 12 or older and each dose is about 60ug. In some such embodiments, an RNA (e.g., mRNA) composition describedherein is administered to subjects of age 12 or older and each dose isabout 50 ug. In one embodiment, the paediatric population comprises orconsists of subjects 12 to less than 18 years of age including subjects16 to less than 18 years of age and/or subjects 12 to less than 16 yearsof age. In this embodiment, treatments may comprise 2 vaccinations 21days apart, wherein, in one embodiment, the vaccine is administered inan amount of 30 μg RNA per dose, e.g., by intramuscular administration.In some embodiments, higher doses are administered to older pediatricpatients and adults, e.g., to patients 12 years or older, compared toyounger children or infants, e.g. 2 to less than 5 years old, 6 monthsto less than 2 years old, or less than 6 months old. In someembodiments, higher doses are administered to children who are 2 to lessthan 5 years old, as compared to toddlers and/or infants, e.g., who are6 months to less than 2 years old, or less than 6 months old.

In one embodiment, the paediatric population comprises or consists ofsubjects 5 to less than 18 years of age including subjects 12 to lessthan 18 years of age and/or subjects 5 to less than 12 years of age. Inthis embodiment, treatments may comprise 2 vaccinations 21 days apart,wherein, in various embodiments, the vaccine is administered in anamount of 10 μg, 20 μg, or 30 μg RNA per dose, e.g., by intramuscularadministration. In some such embodiments, an RNA (e.g., mRNA)composition described herein is administered to subjects of age 5 to 11and each dose is about 10 ug

In some embodiments, each dose comprises about 5 ug of RNA encoding aSARS-CoV-2 S protein of a first variant and about 5 ug of RNA encoding aSARS-CoV-2 S protein of a second variant. In some embodiments, each dosecomprises about 5 ug of RNA encoding a SARS-CoV-2 S protein of a Wuhanstrain and about 5 ug of RNA encoding a SARS-CoV-2 S protein of anOmicron variant. In some embodiments, each dose comprises about 5 ug ofRNA encoding a SARS-CoV-2 S protein of a Wuhan strain (e.g., RNAcomprising SEQ ID NO: 20) and about 5 ug of RNA encoding a SARS-CoV-2 Sprotein of a BA.1 Omicron variant (e.g., RNA comprising SEQ ID NO: 51).In some embodiments, each dose comprises about 5 ug of RNA encoding aSARS-CoV-2 S protein of a Wuhan strain (e.g., RNA comprising SEQ ID NO:20) and about 5 ug of RNA encoding a SARS-CoV-2 S protein of a BA.4/5Omicron variant (e.g., RNA comprising SEQ ID NO: 72).

In one embodiment, the paediatric population comprises or consists ofsubjects less than 5 years of age including subjects 2 to less than 5years of age, subjects 12 to less than 24 months of age, subjects 7 toless than 12 months of age, subjects 6 to less than 12 months of ageand/or subjects less than 6 months of age. In this embodiment,treatments may comprise 2 vaccinations, e.g., 21 to 42 days apart, e.g.,21 days apart, wherein, in various embodiments, the vaccine isadministered in an amount of 3 μg, 10 μg, 20 μg, or 30 μg RNA per dose,e.g., by intramuscular administration. In some such embodiments, an RNA(e.g., mRNA) composition described herein is administered to subjects ofage 2 to less than 5 and each dose is about 3 ug. In some suchembodiments, an RNA (e.g., mRNA) composition described herein isadministered to subjects of about 6 months to less than about 5 yearsand each dose is about 3 ug.

In some embodiments, each dose comprises about 1.5 ug of RNA encoding aSARS-CoV-2 S protein of a first variant and about 1.5 ug of RNA encodinga SARS-CoV-2 S protein of a second variant. In some embodiments, eachdose comprises about 1.5 ug of RNA encoding a SARS-CoV-2 S protein of aWuhan strain and about 1.5 ug of RNA encoding a SARS-CoV-2 S protein ofan Omicron variant. In some embodiments, each dose comprises about 1.5ug of RNA encoding a SARS-CoV-2 S protein of a Wuhan strain (e.g., RNAcomprising SEQ ID NO: 20) and about 1.5 ug of RNA encoding a SARS-CoV-2S protein of a BA.1 Omicron variant (e.g., RNA comprising SEQ ID NO:51). In some embodiments, each dose comprises about 1.5 ug of RNAencoding a SARS-CoV-2 S protein of a Wuhan strain (e.g., RNA comprisingSEQ ID NO: 20) and about 1.5 ug of RNA encoding a SARS-CoV-2 S proteinof a BA.4/5 Omicron variant (e.g., RNA comprising SEQ ID NO: 72).

In some embodiments, an RNA (e.g., mRNA) composition described herein isadministered to subjects of age 12 or older and at least one dose givenin a vaccination regimen (e.g., a primary vaccination regimen and/or abooster vaccination regimen) is about 60 ug. In some embodiments, an RNA(e.g., mRNA) composition described herein is administered to subjects ofage 12 or older and at least one dose given in a vaccination regimen(e.g., a primary vaccination regimen and/or a booster vaccinationregimen) is about 30 ug. In some embodiments, an RNA (e.g., mRNA)composition described herein is administered to subjects of age 12 orolder and at least one dose given in a vaccination regimen (e.g., aprimary vaccination regimen and/or a booster vaccination regimen) isabout 15 ug. In some embodiments, an RNA (e.g., mRNA) compositiondescribed herein is administered to subjects of age 5 to less than 12years of age and at least one dose given in a vaccination regimen (e.g.,a primary vaccination regimen and/or a booster vaccination regimen) isabout 10 ug. In some embodiments, an RNA (e.g., mRNA) compositiondescribed herein is administered to subjects of age 2 to less than 5 andat least one dose given in a vaccination regimen (e.g., a primaryvaccination regimen and/or a booster vaccination regimen) is about 3 ug.In some embodiments, an RNA (e.g., mRNA) composition described herein isadministered to subjects of 6 months to less than age 2 and at least onedose given in a vaccination regimen (e.g., a primary vaccination regimenand/or a booster vaccination regimen) is about 3 ug or lower, including,e.g., 2 ug, 1 ug, or lower). In some embodiments, an RNA (e.g., mRNA)composition described herein is administered to infants of less than 6months and at least one dose given in a vaccination regimen (e.g., aprimary vaccination regimen and/or a booster vaccination regimen) isabout 3 ug or lower, including, e.g., 2 ug, 1 ug, 0.5 ug, or lower).

In some embodiments, an RNA (e.g., mRNA) composition described herein isadministered (e.g., by intramuscular injection) as a single dose. Insome embodiments, a single dose comprise a single RNA encoding aSARS-CoV-2 S protein or an immunogenic fragment thereof (e.g., an RBDdomain). In some embodiments, a single dose comprise at least two RNAsdescribed herein, for example, each RNA encoding a SARS-CoV-2 S proteinor an immunogenic fragment thereof (e.g., an RBD domain) from differentstrains. In some embodiments, such at least two RNAs described hereincan be administered as a single mixture. For example, in some suchembodiments, two separate RNA compositions described herein can be mixedto generate a single mixture prior to injection. In some embodiments,such at least two RNAs described herein can be administered as twoseparate compositions, which, for example, can be administered atdifferent injection sites (e.g., on different arms, or different siteson the same arm).

In some embodiments, a dose administered to subjects in need thereof maycomprise administration of a single RNA (e.g., mRNA) compositiondescribed herein.

In some embodiments, a dose administered to subjects in need thereof maycomprise administration of at least two or more (including, e.g., atleast three or more) different drug products/formulations. For example,in some embodiments, at least two or more different drugproducts/formulations may comprise at least two different RNA (e.g.,mRNA) compositions described herein (e.g., in some embodiments eachcomprising a different RNA construct).

In some embodiments, an RNA (e.g., mRNA) composition disclosed hereinmay be administered in conjunction with a vaccine targeting a differentinfectious agent. In some embodiments, the different infectious agent isone that increases the likelihood of a subject experiencing deleterioussymptoms when coinfected with SARS-CoV-2 and the infectious agent. Insome embodiments, the infectious agent is one that increases theinfectivity of SARS-CoV-2 when a subject is coinfected with SARS-CoV-2and the infectious agent. In some embodiments, at least one RNA (e.g.,mRNA) composition described herein may be administered in combinationwith a vaccine that targets influenza. In some embodiments, at least twoor more different drug products/formulations may comprise at least oneRNA (e.g., mRNA) composition described herein and a vaccine targeting adifferent infectious agent (e.g., an influenza vaccine). In someembodiments, different drug products/formulations are separatelyadministered. In some embodiments, such different drugproduct/formulations are separately administered at the same time (e.g.,at the same vaccination session) at different sites of a subject (e.g.,at different arms of the subject).

In one embodiment, at least two doses are administered. For example, asecond dose may be administered about 21 days following administrationof the first dose.

In some embodiments, at least one single dose is administered. In someembodiments, such single dose is administered to subjects, for example,who may have previously received one or more doses of, or a completeregimen of, a SARS-CoV-2 vaccine (e.g., of a BNT162b2 vaccine[including, e.g., as described herein], an mRNA-1273 vaccine, anAd26.CoV2.S vaccine, a ChAdxOx1 vaccine, an NVX-CoV2373 vaccine, aCvnCoV vaccine, a GAM-COVID0Vac vaccine, a CoronaVac vaccine, aBBIBP-CorV vaccine, an Ad5-nCoV vaccine, a zf2001 vaccine, a SCB-2019vaccine, or other approved RNA (e.g., mRNA) or adenovector vaccines,etc. Alternatively or additionally, in some embodiments, a single doseis administered to subjects who have been exposed to and/or infected bySARS-CoV-2. In some embodiments, at least one single dose isadministered to subjects who both have received one or more doses of, ora complete regimen of, a SARS-CoV-2 vaccine and have been exposed toand/or infected with SARS-CoV-2.

In some particular embodiments where at least one single dose isadministered to subjects who have received one or more doses of a priorSARS-CoV-2 vaccine, such prior SARS-CoV-2 vaccine is a differentvaccine, or a different form (e.g., formulation) and/or dose of avaccine with the same active (e.g., BNT162b2); in some such embodiments,such subjects have not received a complete regimen of such prior vaccineand/or have experienced one or more undesirable reactions to or effectsof one or more received doses of such prior vaccine. In some particularembodiments, such prior vaccine is or comprises higher dose(s) of thesame active (e.g., BNT162b2). Alternatively or additionally, in somesuch embodiments, such subjects were exposed to and/or infected bySARS-CoV-2 prior to completion (but, in some embodiments, afterinitiation) of a full regimen of such prior vaccine.

In one embodiment, at least two doses are administered. For example, asecond dose may be administered about 21 days following administrationof the first dose.

In one embodiment, at least three doses are administered. In someembodiments, such third dose is administered a period of time after thesecond dose that is comparable to (e.g., the same as) the period of timebetween the first and second doses. For example, in some embodiments, athird dose may be administered about 21 days following administration ofthe second dose. In some embodiments, a third dose is administered aftera longer period of time relative to the second dose than the second dosewas relative to the first dose. In some embodiments, a three-doseregimen is administered to an immunocompromised patient, e.g., a cancerpatient, an HIV patient, a patient who has received and/or is receivingimmunosuppressant therapy (e.g., an organ transplant patient). In someembodiments, the length of time between the second and third dose (e.g.,a second and third dose administered to an immunocompromised patient) isat least about 21 days (e.g., at least about 28 days).

In some embodiments, a vaccination regimen comprises administering thesame amount of RNA in different doses (e.g., in first and/or secondand/or third and/or subsequent doses). In some embodiments, avaccination regimen comprises administering different amounts of RNA indifferent doses. In some embodiments, one or more later doses is largerthan one or more earlier doses (e.g., in situations where waning ofvaccine efficacy from one or more earlier doses is observed and/orimmune escape by a variant (e.g., one described herein) that isprevalent or rapidly spreading is observed in a relevant jurisdiction atthe time of administration is observed). In some embodiments, one ormore later doses may be larger than one or more earlier doses by atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, or higher, provided that safetyand/or tolerability of such a dose is clinically acceptable. In someembodiments, one or more later doses may be larger than one or moreearlier doses by at least 1.1-fold, at least 1.5-fold, at least 2-fold,at least 3-fold, at least 4-fold, or higher provided that safety and/ortolerability of such a dose is clinically acceptable. In someembodiments, one or more later doses is smaller than one or more earlierdoses (e.g., in a negative reaction was experienced after one or moreearlier doses and/or if exposure to and/or infection by SARS-CoV-2between an earlier dose and a subsequent dose). In some embodiments, oneor more later doses may be smaller than one or more earlier doses by atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, or higher. In some embodiments, where different doses areutilized, they are related to one another by identity with and/ordilution of a common stock as described herein. In some embodiments,where at least two or more doses are administered (e.g., at least twodoses administered in a primary regimen, at least two doses administeredin a booster regimen, or at least one dose administered in a primaryregimen and at least one dose in a booster regimen), the same RNAcompositions described herein may be administered in such doses and eachof such doses can be the same or different (as described herein). Insome embodiments, where at least two or more doses are administered(e.g., at least two doses administered in a primary regimen, at leasttwo doses administered in a booster regimen, or at least one doseadministered in a primary regimen and at least one dose in a boosterregimen), different RNA compositions described herein (e.g., differentencoded viral polypeptides, e.g., from different coronavirus clades, orfrom different strains of the same coronavirus clade; differentconstruct elements such as 5′ cap, 3′ UTR, 5′ UTR, etc.; differentformulations, e.g., different excipients and/or buffers (e.g., PBS vs.Tris); different LNP compositions; or combinations thereof) may beadministered in such doses and each of such doses can be the same ordifferent (e.g., as described herein).

In some embodiments, a subject is administered two or more RNAs (e.g.,as part of either a primary regimen or a booster regimen), wherein thetwo or more RNAs are administered on the same day or same visit. In someembodiments, the two or more RNAs are administered in separatecompositions, e.g., by administering each RNA to a separate part of thesubject (e.g., by intramuscular administration to different arms of thesubject or to different sites of the same arm of the subject). In someembodiments, the two or more RNAs are mixed prior to administration(e.g., mixed immediately prior to administration, e.g., by theadministering practitioner). In some embodiments, the two or more RNAsare formulated together (e.g., by (a) mixing separate populations ofLNPs, each population comprising a different RNA; or (b) by mixing twoor more RNAs prior to LNP formulation, so that each LNP comprises two ormore RNAs). In some embodiments, the two or more RNAs comprise an RNAthat encode a coronavirus S protein or immunogenic fragment thereof(e.g., RBD or other relevant domains) from one strain (e.g., Wuhanstrain) and a variant that is prevalent or rapidly spreading in arelevant jurisdiction at the time of administration (e.g., a variantdescribed herein). In some embodiments, such a variant is an Omicronvariant (e.g., a BA.1, BA.2, or BA.3 variant). In some embodiments, thetwo or more RNAs comprise a first RNA and a second RNA that have beenshown to elicit a broad immune response in subject. In some embodimentsthe two or more RNAs comprise an RNA encoding a SARS-CoV-2 S proteinfrom a Wuhan strain and an RNA encoding a SARS-CoV-2 S protein from aBA.1 Omicron variant. In some embodiments the two or more RNAs comprisean RNA encoding a SARS-CoV-2 S protein from a Wuhan strain and an RNAencoding a SARS-CoV-2 S protein from a BA.2 Omicron variant. In someembodiments the two or more RNAs comprise an RNA encoding a SARS-CoV-2 Sprotein from a Wuhan strain and an RNA encoding a SARS-CoV-2 S proteinfrom a BA.4 or BA.5 Omicron variant. In some embodiments the two or moreRNAs comprise an RNA encoding a SARS-CoV-2 S protein from a BA.1 Omicronvariant and an RNA encoding a SARS-CoV-2 S protein from a BA.2 Omicronvariant. In some embodiments the two or more RNAs comprise an RNAencoding a SARS-CoV-2 S protein from a BA.1 Omicron variant and an RNAencoding a SARS-CoV-2 S protein from a BA.4 or BA.5 Omicron variant. Insome embodiments the two or more RNAs comprise an RNA encoding aSARS-CoV-2 S protein from a BA.2 Omicron variant and an RNA encoding aSARS-CoV-2 S protein from a BA.4 or 5 Omicron variant. In someembodiments the two or more RNAs comprise an RNA encoding a SARS-CoV-2 Sprotein from a Wuhan strain, an alpha variant, a beta variant, or adelta variant, or sublineages derived therefrom; and an RNA encoding aSARS-CoV-2 S protein from a BA.2, BA.4 or 5 Omicron variant, orsublineages derived therefrom.

In some embodiments, a subject may be administered any one ofcombinations 1 to 66, listed in the below table. In some embodiments,such combinations can be administered using an LNP formulation, wherethe first RNA and the second RNA are encapsulated in the same LNP or inseparate LNPs. In some embodiments, such combinations can beadministered as separate LNP formulations (e.g., by administering atseparate sites to a subject).

SARS-CoV-2 S SARS-CoV-2 S protein encoded protein encoded Combination bya first RNA¹ by a second RNA¹ 1 Wuhan Alpha 2 Wuhan Beta 3 Wuhan Delta 4Wuhan BA.1 5 Wuhan BA.2 6 Wuhan BA.2.12.1 7 Wuhan BA.3 8 Wuhan BA.4/5 9Wuhan XBB 10 Wuhan XBB variant (e.g., XBB.1, XBB.2, XBB.1.3) 11 WuhanBQ.1.1 12 Alpha Beta 13 Alpha Delta 14 Alpha BA.1 15 Alpha BA.2 16 AlphaBA.2.12.1 17 Alpha BA.3 18 Alpha BA.4/5 19 Alpha XBB 20 Alpha XBBvariant (e.g., XBB.1, XBB.2, XBB.1.3) 21 Alpha BQ.1.1 22 Beta Delta 23Beta BA.1 24 Beta BA.2 25 Beta BA.2.12.1 26 Beta BA.3 27 Beta BA.4/5 28Beta XBB 29 Beta XBB variant (e.g., XBB.1, XBB.2, XBB.1.3) 30 BetaBQ.1.1 31 Delta BA.1 32 Delta BA.2 33 Delta BA.2.12.1 34 Delta BA.3 35Delta BA.4/5 36 Delta XBB 37 Delta XBB variant (e.g., XBB.1, XBB.2,XBB.1.3) 38 Delta BQ.1.1 39 BA.1 BA.2 40 BA.1 BA.2.12.1 41 BA.1 BA.3 42BA.1 BA.4/5 43 BA.1 XBB 44 BA.1 XBB variant (e.g., XBB.1, XBB.2,XBB.1.3) 45 BA.1 BQ.1.1 46 BA.2 BA.2.12.1 47 BA.2 BA.3 48 BA.2 BA.4/5 49BA.2 XBB 50 BA.2 XBB variant (e.g., XBB.1, XBB.2, XBB.1.3) 51 BA.2BQ.1.1 52 BA.2.12.1 BA.3 53 BA.2.12.1 BA.4/5 54 BA.2.12.1 XBB 55BA.2.12.1 XBB variant (e.g., XBB.1, XBB.2, XBB.1.3) 56 BA.2.12.1 BQ.1.157 BA.3 BA.4/5 58 BA.3 XBB 59 BA.3 XBB variant (e.g., XBB.1, XBB.2,XBB.3) 60 BA.3 BQ.1.1 61 BA.4/5 XBB 62 BA.4/5 XBB variant (e.g., XBB.1,XBB.2, XBB.1.3) 63 BA.4/5 BQ.1.1 64 XBB XBB variant (e.g., XBB.1, XBB.2,XBB.1.3) 65 XBB BQ.1.1 66 XBB variant (e.g., BQ.1.1 XBB.1, XBB.2,XBB.1.3) ¹Listed RNAs encode a SARS-CoV-2 S protein having mutationscharacteristic of the indicated SARS-CoV-2 variant.

In some embodiments, a subject is administered a first RNA and a secondRNA, each in the same amount (i.e., at a 1:1 ratio).

In some embodiments, a subject is administered a first RNA and a secondRNA, each in a different amount. For example, in some embodiments, asubject is administered a first RNA in an amount that is 0.01 to 100times that of a second RNA (e.g., wherein the amount of a first RNA is0.01 to 50, 0.01 to 4, 0.01 to 30, 0.01 to 25, 0.01 to 20, 0.01 to 15,0.01 to 10, 0.01 to 9, 0.01 to 8, 0.01 to 7, 0.01 to 6, 0.01 to 5, 0.01to 4, 0.01 to 3, 0.01 to 2, 0.01 to 1.5, 1 to 50, 1 to 4, 1 to 30, 1 to25, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1to 4, 1 to 3, 1 to 2, or 1 to 1.5 times that of a second RNA). In someembodiments, a subject is administered a first RNA and a second RNA,wherein the concentration of the first RNA is 1 to 10 times that of thesecond RNA. In some embodiments, a subject is administered a first RNAand a second RNA, wherein the amount of the first RNA is 1 to 5 timesthat of the second RNA. In some embodiments, a subject is administered afirst RNA and a second RNA, wherein the concentration of the first RNAis 1 to 3 times that of the second RNA. In some embodiments, a subjectis administered a first RNA and a second RNA, wherein the amount of thefirst RNA is 2 times that of the second RNA. In some embodiments, asubject is administered a first RNA and a second RNA, wherein theconcentration of the first RNA is 3 times that of the second RNA.

In some embodiments, a subject is administered three RNAs, each encodinga SARS-CoV-2 S protein comprising one or more mutations characteristicof a different SARS-CoV-2 variant, and each in the same amount (i.e., ata 1:1:1 ratio).

In some embodiments, a subject is administered three RNAs, each encodinga SARS-CoV-2 S protein comprising one or more mutations characteristicof a different SARS-CoV-2 variant, wherein the amount of each RNA is notthe same (e.g., one RNA is present in an amount that is different thanthe other two RNA, or all three RNAs are present in different amounts).For example, in some embodiments, the ratio of first RNA:secondRNA:third RNA is 1:0.01-100: 0.01-100 (e.g., 1:0.01-50:0.01-50;1:0.01-40:0.01-40; 1:0.01-30:0.01-25; 1:0.01-25:0.01-25;1:0.01-20:0.01-20; 1:0.01-15:0.01-15; 1:0.01-10:0.01-9; 1:0.01-9:0.01-9;1:0.01-8:0.01-8; 1:0.01-7:0.01-7; 1:0.01-6:0.01-6; 1:0.01-5:0.01-5;1:0.01-4:0.01-4; 1:0.01-3:0.01-3; 1:0.01-2:0.01-2; or1:0.01-1.5:0.01-1.5). In some embodiments, a subject is administeredthree RNAs at a ratio of 1:1:3. In some embodiments, a subject isadministered three RNAs at a ratio of 1:3:3.

In some embodiments, a vaccination regimen comprises a first vaccinationregimen (e.g., a primary regimen) that includes at least two doses of anRNA composition as described herein, e.g., wherein the second dose maybe administered about 21 days following administration of the firstdose, and a second vaccination (e.g., a booster regimen) that comprisesa single dose or multiple doses, e.g., two doses, of an RNA compositionas described herein. In some embodiments, doses of a booster regimen arerelated to those of a primary regimen by identity with or dilution froma common stock as described herein. In various embodiments, a boosterregimen is administered (e.g., is initiated) at least 1 week, at least 2weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3months, at least 4 months, at least 5 months, or at least 6 months, atleast 7 months, at least 8 months, at least 9 months, at least 10months, at least 11 months, at least 12 months, or longer, afteradministration of a primary regimen, e.g., after completion of a primaryregimen comprising at least two doses. In various embodiments, a boosterregimen is administered (e.g., is initiated) 1-12 months, 2-12 months,3-12 months, 4-12 months, 6-12 months, 1-6 months, 1-5 months, 1-4months, 1-3 months, or 2-3 months after administration of a primaryregimen, e.g., after completion of a primary regimen comprising at leasttwo doses. In various embodiments, a booster regimen is administered(e.g., is initiated) 1 to 60 months, 2 to 48 months, 2 to 24 months, 3to 24 months, 6 to 18 months, 6 to 12 months, or 5 to 7 months afteradministration of a primary regimen, e.g., after completion of atwo-dose primary regimen. In some embodiments, each dose of a primaryregimen is about 60 μg per dose. In some embodiments, each dose of aprimary regimen is about 50 μg per dose. In some embodiments, each doseof a primary regimen is about 30 μg per dose. In some embodiments, eachdose of a primary regimen is about 25 μg per dose. In some embodiments,each dose of a primary regimen is about 20 μg per dose. In someembodiments, each dose of a primary regimen is about 15 μg per dose. Insome embodiments, each dose of a primary regimen is about 10 ug perdose. In some embodiments, each dose of a primary regimen is about 3 μgper dose. In some embodiments, each dose of a booster regimen is thesame as that of the primary regimen. In some embodiments, each dose of abooster regimen comprises the same amount of RNA as a dose administeredin a primary regimen. In some embodiments, at least one dose of abooster regimen is the same as that of the primary regimen. In someembodiments, at least one dose of a booster regimen comprises the sameamount of RNA as at least one dose of a primary regimen. In someembodiments, at least one dose of a booster regimen is lower than thatof the primary regimen. In some embodiments, at least one dose of abooster regimen comprises an amount of RNA that is lower than that of aprimary regimen. In some embodiments, at least one dose of a boosterregimen is higher than that of the primary regimen. In some embodiments,at least one dose of a booster regimen comprises an amount of RNA thatis higher than that of a primary regimen.

In some embodiments, a booster regimen (e.g., as described herein) isadministered to a pediatric patient (e.g., a patient aged 2 through 5years old, a patient aged 5 through 11 years old, or a patient aged 12through 15 years old). In some embodiments, a booster regimen isadministered to a pediatric patient who is 6 months old to less than 2years old. In some embodiments, a booster regimen is administered to apediatric patient who is less than 6 months old. In some embodiments, abooster regimen is administered to a pediatric patient who is 6 monthsold to less than 5 years old. In some embodiments, a booster regimen isadministered to a pediatric patient who is 2 years old to less than 5years old. In some embodiments, a booster regimen is administered to apediatric patient who is 5 years old to less than 12 years old. In someembodiments, a booster regimen is administered to a pediatric patientwho is 12 years old to less than 16 years old. In some embodiments, eachdose of a pediatric booster regimen comprises about 3 μg of RNA. In someembodiments, each dose of a pediatric booster regimen comprises about 10μg of RNA. In some embodiments, each dose of a pediatric booster regimencomprises about 15 μg of RNA. In some embodiments, each dose of apediatric booster regimen comprises about 20 μg of RNA. In someembodiments, each dose of a pediatric booster regimen comprises about 25μg of RNA. In some embodiments, each dose of a pediatric booster regimencomprises about 30 μg of RNA. In some embodiments, a booster regimen isadministered to a non-pediatric patient (e.g., a patient 16 years orolder, a patient aged 18 through 64 years old, and/or a patient 65 yearsand older). In some embodiments, each dose of a non-pediatric boosterregimen comprises about 3 ug of RNA, about 10 ug of RNA, about 25 μg orRNA, about 30 μg of RNA, about 40 μg of RNA, about 45 μg of RNA, about50 μg of RNA, about 55 μg of RNA, or about 60 μg of RNA. In someembodiments, the same booster regimen may be administered to bothpediatric and non-pediatric patients (e.g., to a patient 12 years orolder). In some embodiments, a booster regimen that is administered to anon-pediatric patient is administered in a formulation and dose that isrelated to that of a primary regimen previously received by the patientby identity with or by dilution as described herein. In someembodiments, a non-pediatric patient who receives a booster regimen at alower dose than a primary regimen may have experienced an adversereaction to one or more doses of such primary regimen and/or may havebeen exposed to and/or infected by SARS-CoV-2 between such primaryregimen and such booster regimen, or between doses of such primaryregimen and/or of such booster regimen. In some embodiments, pediatricand non-pediatric patients may receive a booster regimen at a higherdose than a primary regimen when waning of vaccine efficacy at lowerdoses is observed, and/or when immune escape of a variant that isprevalent and/or spreading rapidly at a relevant jurisdiction at thetime of administration is observed.

In some embodiments one or more doses of a booster regimen differs fromthat of a primary regimen. For example, in some embodiments, anadministered dose may correspond to a subject's age and a patient mayage out of one treatment age group and into a next. Alternatively oradditionally, in some embodiments, an administered dose may correspondto a patient's condition (e.g., immunocompromised state) and a differentdose may be selected for one or more doses of a booster regimen than fora primary regimen (e.g., due to intervening cancer treatment, infectionwith HIV, receipt of immunosuppressive therapy, for example associatedwith an organ transplant. In some embodiments, at least one dose of abooster regimen may comprise an amount of RNA that is higher than atleast one dose administered in a primary regimen (e.g., in situationswhere waning of vaccine efficacy from one or more earlier doses isobserved and/or immune escape by a variant (e.g., one described herein)that is prevalent or rapidly spreading is observed in a relevantjurisdiction at the time of administration).

In some embodiments, a primary regimen may involve one or more 3 ugdoses and a booster regimen may involve one or more 10 ug doses, and/orone or more 20 ug doses, or one or more 30 ug doses. In someembodiments, a primary regimen may involve one or more 3 ug doses and abooster regimen may involve one or more 3 ug doses. In some embodiments,a primary regimen may involve two or more 3 ug doses (e.g., at least twodoses, each comprising 3 ug of RNA, and administered about 21 days afterone another) and a booster regimen may involve one or more 3 ug doses.In some embodiments, a primary regimen may involve one or more 10 ugdoses and a booster regimen may involve one or more 20 ug doses, and/orone or more 30 ug doses. In some embodiments, a primary regimen mayinvolve one or more 10 ug doses and a booster regimen may involve one ormore 10 ug doses. In some embodiments, a primary regimen may involve twoor more 10 ug doses (e.g., two doses, each comprising 10 ug of RNA,administered about 21 days apart) and a booster regimen may involve oneor more 10 ug doses. In some embodiments, a primary regimen may involveone or more 20 ug doses and a booster regimen may involve one or more 30ug doses. In some embodiments, a primary regimen may involve one or more20 ug doses and a booster regimen may involve one or more 20 ug doses.In some embodiments, a primary regimen may involve one or more 30 ugdoses, and a booster regimen may also involve one or more 30 ug doses.In some embodiments, a primary regimen may involve two or more 30 ugdoses (e.g., two doses, each comprising 30 ug of RNA, administered about21 days apart), and a booster regimen may also involve one or more 30 ugdoses. In some embodiments, a primary regimen may involve two or more 30ug doses (e.g., two doses, each comprising 30 ug of RNA, administeredabout 21 days apart), and a booster regimen may involve one or more 50ug doses. In some embodiments, a primary regimen may involve two or more30 ug doses (e.g., two doses, each comprising 30 ug of RNA, administeredabout 21 days apart), and a booster regimen may involve one or more 60ug doses.

In some embodiments, a subject is administered a booster regimencomprising at least one 30 ug dose of RNA. In some embodiments, asubject is administered a booster regimen comprising at least one 30 ugdose of RNA encoding a SARS-CoV-2 S protein from a Wuhan strain ofSARS-CoV-2 (e.g., BNT162b2). In some embodiments, a subject isadministered a booster regimen comprising at least one dose of 30 ug ofRNA encoding a SARS-CoV-2 S protein having one or more mutations thatare characteristic of a SARS-CoV-2 variant (e.g., a variant describedherein). In some embodiments, a subject is administered a boosterregimen comprising at least one dose of 30 ug of RNA encoding aSARS-CoV-2 S protein having one or more mutations that arecharacteristic of an Omicron variant (e.g., a BA.1, BA.2, BA.3, or BA.4or BA.5 Omicron variant). In some embodiments, a subject is administereda booster regimen comprising at least one dose of 30 ug of RNA, whereinthe 30 ug of RNA comprises RNA encoding a SARS-CoV-2 S protein from aWuhan strain and RNA encoding a SARS-CoV-2 S protein comprisingmutations that are characteristic of a SARS-CoV-2 variant (e.g., in someembodiments, a subject is administered a booster regimen comprising atleast one dose comprising 15 ug of RNA encoding a SARS-CoV-2 S proteinfrom a Wuhan strain and 15 ug of RNA encoding a SARS-CoV-2 S proteinhaving one or more mutations that are characteristic of an Omicronvariant). In some embodiments, a subject is administered a boosterregimen comprising at least one dose comprising 15 ug of RNA encoding aSARS-CoV-2 S protein from a Wuhan strain and 15 ug of RNA encoding aSARS-CoV-2 S protein having one or more mutations that arecharacteristic of a BA.1 Omicron variant. In some embodiments, a subjectis administered a booster regimen comprising at least one dosecomprising 10 ug of RNA encoding a SARS-CoV-2 S protein from a Wuhanstrain and 20 ug of RNA encoding a SARS-CoV-2 S protein having one ormore mutations that are characteristic of a BA.1 Omicron variant. Insome embodiments, a subject is administered a booster regimen comprisingat least one dose comprising 7.5 ug of RNA encoding a SARS-CoV-2 Sprotein from a Wuhan strain and 22.5 ug of RNA encoding a SARS-CoV-2 Sprotein having one or more mutations that are characteristic of a BA.1Omicron variant. In some embodiments, a subject is administered abooster regimen comprising at least one dose comprising 15 ug of RNAencoding a SARS-CoV-2 S protein from a Wuhan strain and 15 ug of RNAencoding a SARS-CoV-2 S protein having one or more mutations that arecharacteristic of a BA.2 Omicron variant. In some embodiments, a subjectis administered a booster regimen comprising at least one dosecomprising 10 ug of RNA encoding a SARS-CoV-2 S protein from a Wuhanstrain and 20 ug of RNA encoding a SARS-CoV-2 S protein having one ormore mutations that are characteristic of a BA.2 Omicron variant. Insome embodiments, a subject is administered a booster regimen comprisingat least one dose comprising 7.5 ug of RNA encoding a SARS-CoV-2 Sprotein from a Wuhan strain and 22.5 ug of RNA encoding a SARS-CoV-2 Sprotein having one or more mutations that are characteristic of a BA.2Omicron variant. In some embodiments, a subject is administered abooster regimen comprising at least one dose comprising 15 ug of RNAencoding a SARS-CoV-2 S protein from a Wuhan strain and 15 ug of RNAencoding a SARS-CoV-2 S protein having one or more mutations that arecharacteristic of a BA.3 Omicron variant. In some embodiments, a subjectis administered a booster regimen comprising at least one dosecomprising 10 ug of RNA encoding a SARS-CoV-2 S protein from a Wuhanstrain and 20 ug of RNA encoding a SARS-CoV-2 S protein having one ormore mutations that are characteristic of a BA.3 Omicron variant. Insome embodiments, a subject is administered a booster regimen comprisingat least one dose comprising 7.5 ug of RNA encoding a SARS-CoV-2 Sprotein from a Wuhan strain and 22.5 ug of RNA encoding a SARS-CoV-2 Sprotein having one or more mutations that are characteristic of a BA.3Omicron variant. In some embodiments, a subject is administered abooster regimen comprising at least one dose comprising 15 ug of RNAencoding a SARS-CoV-2 S protein from a Wuhan strain and 15 ug of RNAencoding a SARS-CoV-2 S protein having one or more mutations that arecharacteristic of a BA.4 or BA.5 Omicron variant. In some embodiments, asubject is administered a booster regimen comprising at least one dosecomprising 10 ug of RNA encoding a SARS-CoV-2 S protein from a Wuhanstrain and 20 ug of RNA encoding a SARS-CoV-2 S protein having one ormore mutations that are characteristic of a BA.4 or BA.5 Omicronvariant. In some embodiments, a subject is administered a boosterregimen comprising at least one dose comprising 7.5 ug of RNA encoding aSARS-CoV-2 S protein from a Wuhan strain and 22.5 ug of RNA encoding aSARS-CoV-2 S protein having one or more mutations that arecharacteristic of a BA.4 or BA.5 Omicron variant.

In some embodiments, a subject is administered a booster regimencomprising at least one dose comprising 15 ug of RNA encoding aSARS-CoV-2 S protein from a BA.1 Omicron variant and 15 ug of RNAencoding a SARS-CoV-2 S protein having one or more mutations that arecharacteristic of a BA.2 Omicron variant. In some embodiments, a subjectis administered a booster regimen comprising at least one dosecomprising 10 ug of RNA encoding a SARS-CoV-2 S protein from a BA.1Omicron variant and 20 ug of RNA encoding a SARS-CoV-2 S protein havingone or more mutations that are characteristic of a BA.2 Omicron variant.In some embodiments, a subject is administered a booster regimencomprising at least one dose comprising 7.5 ug of RNA encoding aSARS-CoV-2 S protein from a BA.1 Omicron variant and 22.5 ug of RNAencoding a SARS-CoV-2 S protein having one or more mutations that arecharacteristic of a BA.2 Omicron variant. In some embodiments, a subjectis administered a booster regimen comprising at least one dosecomprising 15 ug of RNA encoding a SARS-CoV-2 S protein from a BA.1Omicron variant and 15 ug of RNA encoding a SARS-CoV-2 S protein havingone or more mutations that are characteristic of a BA.3 Omicron variant.In some embodiments, a subject is administered a booster regimencomprising at least one dose comprising 10 ug of RNA encoding aSARS-CoV-2 S protein from a BA.1 Omicron variant and 20 ug of RNAencoding a SARS-CoV-2 S protein having one or more mutations that arecharacteristic of a BA.3 Omicron variant. In some embodiments, a subjectis administered a booster regimen comprising at least one dosecomprising 7.5 ug of RNA encoding a SARS-CoV-2 S protein from a BA.1Omicron variant and 22.5 ug of RNA encoding a SARS-CoV-2 S proteinhaving one or more mutations that are characteristic of a BA.3 Omicronvariant. In some embodiments, a subject is administered a boosterregimen comprising at least one dose comprising 15 ug of RNA encoding aSARS-CoV-2 S protein from a BA.1 Omicron variant and 15 ug of RNAencoding a SARS-CoV-2 S protein having one or more mutations that arecharacteristic of a BA.4 or BA.5 Omicron variant. In some embodiments, asubject is administered a booster regimen comprising at least one dosecomprising 10 ug of RNA encoding a SARS-CoV-2 S protein from a BA.1Omicron variant and 20 ug of RNA encoding a SARS-CoV-2 S protein havingone or more mutations that are characteristic of a BA.4 or BA.5 Omicronvariant. In some embodiments, a subject is administered a boosterregimen comprising at least one dose comprising 7.5 ug of RNA encoding aSARS-CoV-2 S protein from a BA.1 Omicron variant and 22.5 ug of RNAencoding a SARS-CoV-2 S protein having one or more mutations that arecharacteristic of a BA.4 or BA.5 Omicron variant.

In some embodiments, a subject is administered a booster regimencomprising at least one dose comprising 15 ug of RNA encoding aSARS-CoV-2 S protein from a BA.2 Omicron variant and 15 ug of RNAencoding a SARS-CoV-2 S protein having one or more mutations that arecharacteristic of a BA.4 or BA.5 Omicron variant. In some embodiments, asubject is administered a booster regimen comprising at least one dosecomprising 10 ug of RNA encoding a SARS-CoV-2 S protein from a BA.2Omicron variant and 20 ug of RNA encoding a SARS-CoV-2 S protein havingone or more mutations that are characteristic of a BA.4 or BA.5 Omicronvariant. In some embodiments, a subject is administered a boosterregimen comprising at least one dose comprising 7.5 ug of RNA encoding aSARS-CoV-2 S protein from a BA.2 Omicron variant and 22.5 ug of RNAencoding a SARS-CoV-2 S protein having one or more mutations that arecharacteristic of a BA.4 or BA.5 Omicron variant. In some embodiments, asubject is administered a booster regimen comprising at least one dosecomprising 15 ug of RNA encoding a SARS-CoV-2 S protein from a BA.3Omicron variant and 15 ug of RNA encoding a SARS-CoV-2 S protein havingone or more mutations that are characteristic of a BA.4 or BA.5 Omicronvariant. In some embodiments, a subject is administered a boosterregimen comprising at least one dose comprising 10 ug of RNA encoding aSARS-CoV-2 S protein from a BA.3 Omicron variant and 20 ug of RNAencoding a SARS-CoV-2 S protein having one or more mutations that arecharacteristic of a BA.4 or BA.5 Omicron variant. In some embodiments, asubject is administered a booster regimen comprising at least one dosecomprising 7.5 ug of RNA encoding a SARS-CoV-2 S protein from a BA.3Omicron variant and 22.5 ug of RNA encoding a SARS-CoV-2 S proteinhaving one or more mutations that are characteristic of a BA.4 or BA.5Omicron variant.

In some embodiments, a subject is administered a booster regimencomprising two or more doses of 30 ug of RNA, administered at least twomonths apart from each other. For example, in some embodiments, subjectsare administered a booster regimen comprising two doses of 30 ug of RNAencoding a SARS-CoV-2 S protein having one or more mutations that arecharacteristic of an Omicron variant (e.g., a BA.1, BA.2, or BA.4 orBA.5 Omicron variant).

In some embodiments, a subject is administered (i) a primary regimencomprising at least two 30 ug doses of RNA encoding a SARS-CoV-2 Sprotein from a Wuhan strain, wherein the two doses are administered atleast approximately 21 days apart, and (ii) a booster regimen comprisinga dose of 30 ug of RNA encoding a SARS-CoV-2 S protein having one ormutations that are characteristic of an Omicron variant of SARS-CoV-2(e.g., a BA.1, BA.2, BA.3, BA.4, or BA.5 Omicron variant), wherein thebooster regimen is administered at least two months (including, e.g., atleast three months, at least four months, at least five months, at leastsix months, or more) after completion of the primary regimen. In someembodiments, a subject is administered (i) a primary regimen comprisingat least two 30 ug doses of RNA encoding a SARS-CoV-2 S protein from aWuhan strain, wherein the two doses are administered at leastapproximately 21 days apart, and (ii) a booster regimen comprising a 30ug dose of RNA comprising 15 ug RNA encoding a SARS-CoV-2 S protein froma Wuhan strain and 15 ug of RNA encoding a SARS-CoV-2 S protein havingone or more mutations that are characteristic of an Omicron variant.

In some embodiments, a subject is administered (i) a primary regimencomprising at least two 30 ug doses of RNA encoding a SARS-CoV-2 Sprotein from a Wuhan strain, wherein the two doses are administered atleast approximately 21 days apart, and (ii) a booster regimen comprisinga dose of 30 ug of RNA encoding a SARS-CoV-2 S protein having one ormutations that are characteristic of a BA.1 Omicron variant ofSARS-CoV-2, wherein the booster regimen is administered at least twomonths (including, e.g., at least three months, at least four months, atleast five months, at least six months, or more) after completion of theprimary regimen. In some embodiments, a subject is administered (i) aprimary regimen comprising at least two 30 ug doses of RNA encoding aSARS-CoV-2 S protein from a Wuhan strain, wherein the two doses areadministered at least approximately 21 days apart, and (ii) a boosterregimen comprising a 30 ug dose of RNA comprising 15 ug RNA encoding aSARS-CoV-2 S protein from a Wuhan strain and 15 ug of RNA encoding aSARS-CoV-2 S protein having one or more mutations that arecharacteristic of a BA.1 Omicron variant.

In some embodiments, a subject is administered (i) a primary regimencomprising at least two 30 ug doses of RNA encoding a SARS-CoV-2 Sprotein from a Wuhan strain, wherein the two doses are administered atleast approximately 21 days apart, and (ii) a booster regimen comprisinga dose of 30 ug of RNA encoding a SARS-CoV-2 S protein having one ormutations that are characteristic of a BA.2 Omicron variant ofSARS-CoV-2, wherein the booster regimen is administered at least twomonths (including, e.g., at least three months, at least four months, atleast five months, at least six months, or more) after completion of theprimary regimen. In some embodiments, a subject is administered (i) aprimary regimen comprising at least two 30 ug doses of RNA encoding aSARS-CoV-2 S protein from a Wuhan strain, wherein the two doses areadministered at least approximately 21 days apart, and (ii) a boosterregimen comprising a 30 ug dose of RNA comprising 15 ug RNA encoding aSARS-CoV-2 S protein from a Wuhan strain and 15 ug of RNA encoding aSARS-CoV-2 S protein having one or more mutations that arecharacteristic of a BA.2 Omicron variant.

In some embodiments, a subject is administered (i) a primary regimencomprising at least two 30 ug doses of RNA encoding a SARS-CoV-2 Sprotein from a Wuhan strain, wherein the two doses are administered atleast approximately 21 days apart, and (ii) a booster regimen comprisinga dose of 30 ug of RNA encoding a SARS-CoV-2 S protein having one ormutations that are characteristic of a BA.4 or BA.5 Omicron variant ofSARS-CoV-2, wherein the booster regimen is administered at least twomonths (including, e.g., at least three months, at least four months, atleast five months, at least six months, or more) after completion of theprimary regimen. In some embodiments, a subject is administered (i) aprimary regimen comprising at least two 30 ug doses of RNA encoding aSARS-CoV-2 S protein from a Wuhan strain, wherein the two doses areadministered at least approximately 21 days apart, and (ii) a boosterregimen comprising a 30 ug dose of RNA comprising 15 ug RNA encoding aSARS-CoV-2 S protein from a Wuhan strain and 15 ug of RNA encoding aSARS-CoV-2 S protein having one or more mutations that arecharacteristic of a BA.4 or BA.5 Omicron variant.

In some embodiments, a subject is administered (i) a primary regimencomprising at least two 30 ug doses of RNA encoding a SARS-CoV-2 Sprotein from a Wuhan strain, wherein the two doses are administered atleast approximately 21 days apart, and (ii) a booster regimen comprisinga 30 ug dose of RNA comprising 15 ug RNA encoding a SARS-CoV-2 S proteinfrom a BA.1 Omicron variant and 15 ug of RNA encoding a SARS-CoV-2 Sprotein having one or more mutations that are characteristic of a BA.2Omicron variant.

In some embodiments, a subject is administered (i) a primary regimencomprising at least two 30 ug doses of RNA encoding a SARS-CoV-2 Sprotein from a Wuhan strain, wherein the two doses are administered atleast approximately 21 days apart, and (ii) a booster regimen comprisinga 30 ug dose of RNA comprising 15 ug RNA encoding a SARS-CoV-2 S proteinfrom a BA.1 Omicron variant and 15 ug of RNA encoding a SARS-CoV-2 Sprotein having one or more mutations that are characteristic of a BA.4or BA.5 Omicron variant.

In some embodiments, a subject is administered (i) a primary regimencomprising at least two 30 ug doses of RNA encoding a SARS-CoV-2 Sprotein from a Wuhan strain, wherein the two doses are administered atleast approximately 21 days apart, and (ii) a booster regimen comprisinga 30 ug dose of RNA comprising 15 ug RNA encoding a SARS-CoV-2 S proteinfrom a BA.2 Omicron variant and 15 ug of RNA encoding a SARS-CoV-2 Sprotein having one or more mutations that are characteristic of a BA.4or BA.5 Omicron variant.

In some embodiments, a subject is administered (i) a primary regimencomprising at least two 30 ug doses of RNA encoding a SARS-CoV-2 Sprotein from a Wuhan strain, wherein the two doses are administered atleast approximately 21 days apart, and (ii) a booster regimen comprisingat least one 30 ug dose of RNA encoding a SARS-CoV-2 S protein from anon-BA.1 Omicron variant.

In some embodiments, a subject is administered (i) a primary regimencomprising two 30 ug doses of RNA encoding a SARS-CoV-2 S protein from aWuhan strain, wherein the two doses are administered at leastapproximately 21 days apart, and (ii) a booster regimen comprising atleast two 30 ug doses of RNA encoding a SARS-CoV-2 S protein having oneor more mutations characteristic of an Omicron variant, wherein thebooster regimen is administered at least two months (including, e.g., atleast three months, at least four months, at least five months, at leastsix months, or more) after completion of the primary regimen, and thetwo booster doses are administered at least two months apart from eachother.

In some embodiments, a subject is administered a booster regimencomprising at least one 50 ug dose of RNA. In some embodiments, asubject is administered a booster regimen comprising at least one doseof 50 ug of RNA encoding a SARS-CoV-2 S protein from a Wuhan strain(e.g., BNT162b2). In some embodiments, a subject is administered abooster regimen comprising at least one dose of 50 ug of RNA encoding aSARS-CoV-2 S protein having one or more mutations that arecharacteristic of a SARS-CoV-2 variant (e.g., a variant describedherein). In some embodiments, a subject is administered a boosterregimen comprising at least one dose of 50 ug of RNA encoding aSARS-CoV-2 S protein having one or more mutations that arecharacteristic of an Omicron variant. In some embodiments, a subject isadministered a booster regimen comprising at least one 50 ug dose ofRNA, wherein the 50 ug of RNA comprises RNA encoding a SARS-CoV-2 Sprotein from a Wuhan strain and RNA encoding a SARS-CoV-2 S proteincomprising mutations that are characteristic of a SARS-CoV-2 variant(e.g., in some embodiments, a subject is administered a booster regimencomprising a 50 ug dose of RNA comprising 25 ug of RNA encoding aSARS-CoV-2 S protein from a Wuhan strain and 25 ug of RNA encoding aSARS-CoV-2 S protein having one or more mutations that arecharacteristic of an Omicron variant).

In some embodiments, a subject is administered a booster regimencomprising at least one dose comprising 25 ug of RNA encoding aSARS-CoV-2 S protein from a Wuhan strain and 25 ug of RNA encoding aSARS-CoV-2 S protein having one or more mutations that arecharacteristic of a BA.1 Omicron variant. In some embodiments, a subjectis administered a booster regimen comprising at least one dosecomprising 25 ug of RNA encoding a SARS-CoV-2 S protein from a Wuhanstrain and 25 ug of RNA encoding a SARS-CoV-2 S protein having one ormore mutations that are characteristic of a BA.2 Omicron variant. Insome embodiments, a subject is administered a booster regimen comprisingat least one dose comprising 25 ug of RNA encoding a SARS-CoV-2 Sprotein from a Wuhan strain and 25 ug of RNA encoding a SARS-CoV-2 Sprotein having one or more mutations that are characteristic of a BA.3Omicron variant. In some embodiments, a subject is administered abooster regimen comprising at least one dose comprising 25 ug of RNAencoding a SARS-CoV-2 S protein from a Wuhan strain and 25 ug of RNAencoding a SARS-CoV-2 S protein having one or more mutations that arecharacteristic of a BA.4 or BA.5 Omicron variant.

In some embodiments, a subject is administered a booster regimencomprising at least one dose comprising 25 ug of RNA encoding aSARS-CoV-2 S protein from a BA.1 Omicron variant and 25 ug of RNAencoding a SARS-CoV-2 S protein having one or more mutations that arecharacteristic of a BA.2 Omicron variant. In some embodiments, a subjectis administered a booster regimen comprising at least one dosecomprising 25 ug of RNA encoding a SARS-CoV-2 S protein from a BA.1Omicron variant and 25 ug of RNA encoding a SARS-CoV-2 S protein havingone or more mutations that are characteristic of a BA.3 Omicron variant.In some embodiments, a subject is administered a booster regimencomprising at least one dose comprising 25 ug of RNA encoding aSARS-CoV-2 S protein from a BA.1 Omicron variant and 25 ug of RNAencoding a SARS-CoV-2 S protein having one or more mutations that arecharacteristic of a BA.4 or BA.5 Omicron variant.

In some embodiments, a subject is administered a booster regimencomprising at least one dose comprising 25 ug of RNA encoding aSARS-CoV-2 S protein from a BA.2 Omicron variant and 25 ug of RNAencoding a SARS-CoV-2 S protein having one or more mutations that arecharacteristic of a BA.4 or BA.5 Omicron variant. In some embodiments, asubject is administered a booster regimen comprising at least one dosecomprising 25 ug of RNA encoding a SARS-CoV-2 S protein from a BA.3Omicron variant and 25 ug of RNA encoding a SARS-CoV-2 S protein havingone or more mutations that are characteristic of a BA.4 or BA.5 Omicronvariant.

In some embodiments, a subject is administered (i) a primary regimencomprising two 30 ug doses of RNA encoding a SARS-CoV-2 S protein from aWuhan strain, wherein the two doses are administered approximately 21days apart, and (ii) a booster regimen comprising at least one 50 ugdose of RNA encoding a SARS-CoV-2 S protein having one or mutations thatare characteristic of an Omicron variant of SARS-CoV-2 (e.g., a BA.1,BA.2, BA.4 or BA.5 Omicron variant), wherein the booster regimen isadministered at least two months (including, e.g., at least threemonths, at least four months, at least five months, at least six months,or more) after completion of the primary regimen. In some embodiments, asubject is administered (i) a primary regimen comprising two 30 ug dosesof RNA encoding a SARS-CoV-2 S protein from a Wuhan strain, wherein thetwo doses are administered approximately 21 days apart, and (ii) abooster regimen comprising at least one 50 ug dose of RNA, wherein the50 ug of RNA comprises 25 ug of RNA encoding a SARS-CoV-2 S protein of aWuhan strain and 25 ug of RNA encoding a SARS-CoV-2 S protein having oneor mutations that are characteristic of an Omicron variant (e.g., aBA.1, BA.2, BA.4, or BA.5 variant), wherein the booster regimen isadministered at least two months (including, e.g., at least threemonths, at least four months, at least five months, at least six months,or more) after completion of a first booster regimen. In someembodiments, a subject is administered (i) a primary regimen comprisingat least two 30 ug doses of RNA encoding a SARS-CoV-2 S protein from aWuhan strain, wherein the two doses are administered at leastapproximately 21 days apart, and (ii) a booster regimen comprising adose of 50 ug of RNA encoding a SARS-CoV-2 S protein having one ormutations that are characteristic of a BA.1 Omicron variant ofSARS-CoV-2, wherein the booster regimen is administered at least twomonths (including, e.g., at least three months, at least four months, atleast five months, at least six months, or more) after completion of theprimary regimen. In some embodiments, a subject is administered (i) aprimary regimen comprising at least two 30 ug doses of RNA encoding aSARS-CoV-2 S protein from a Wuhan strain, wherein the two doses areadministered at least approximately 21 days apart, and (ii) a boosterregimen comprising a 50 ug dose of RNA comprising 25 ug RNA encoding aSARS-CoV-2 S protein from a Wuhan strain and 25 ug of RNA encoding aSARS-CoV-2 S protein having one or more mutations that arecharacteristic of a BA.1 Omicron variant.

In some embodiments, a subject is administered (i) a primary regimencomprising at least two 30 ug doses of RNA encoding a SARS-CoV-2 Sprotein from a Wuhan strain, wherein the two doses are administered atleast approximately 21 days apart, and (ii) a booster regimen comprisinga dose of 50 ug of RNA encoding a SARS-CoV-2 S protein having one ormutations that are characteristic of a BA.2 Omicron variant ofSARS-CoV-2, wherein the booster regimen is administered at least twomonths (including, e.g., at least three months, at least four months, atleast five months, at least six months, or more) after completion of theprimary regimen. In some embodiments, a subject is administered (i) aprimary regimen comprising at least two 30 ug doses of RNA encoding aSARS-CoV-2 S protein from a Wuhan strain, wherein the two doses areadministered at least approximately 21 days apart, and (ii) a boosterregimen comprising a 50 ug dose of RNA comprising 25 ug RNA encoding aSARS-CoV-2 S protein from a Wuhan strain and 25 ug of RNA encoding aSARS-CoV-2 S protein having one or more mutations that arecharacteristic of a BA.2 Omicron variant.

In some embodiments, a subject is administered (i) a primary regimencomprising at least two 30 ug doses of RNA encoding a SARS-CoV-2 Sprotein from a Wuhan strain, wherein the two doses are administered atleast approximately 21 days apart, and (ii) a booster regimen comprisinga dose of 50 ug of RNA encoding a SARS-CoV-2 S protein having one ormutations that are characteristic of a BA.4 or BA.5 Omicron variant ofSARS-CoV-2, wherein the booster regimen is administered at least twomonths (including, e.g., at least three months, at least four months, atleast five months, at least six months, or more) after completion of theprimary regimen. In some embodiments, a subject is administered (i) aprimary regimen comprising at least two 30 ug doses of RNA encoding aSARS-CoV-2 S protein from a Wuhan strain, wherein the two doses areadministered at least approximately 21 days apart, and (ii) a boosterregimen comprising a 50 ug dose of RNA comprising 25 ug RNA encoding aSARS-CoV-2 S protein from a Wuhan strain and 25 ug of RNA encoding aSARS-CoV-2 S protein having one or more mutations that arecharacteristic of a BA.4 or BA.5 Omicron variant.

In some embodiments, a subject is administered (i) a primary regimencomprising at least two 30 ug doses of RNA encoding a SARS-CoV-2 Sprotein from a Wuhan strain, wherein the two doses are administered atleast approximately 21 days apart, and (ii) a booster regimen comprisinga 50 ug dose of RNA comprising 25 ug RNA encoding a SARS-CoV-2 S proteinfrom a BA.1 Omicron variant and 25 ug of RNA encoding a SARS-CoV-2 Sprotein having one or more mutations that are characteristic of a BA.2Omicron variant.

In some embodiments, a subject is administered (i) a primary regimencomprising at least two 30 ug doses of RNA encoding a SARS-CoV-2 Sprotein from a Wuhan strain, wherein the two doses are administered atleast approximately 21 days apart, and (ii) a booster regimen comprisinga 50 ug dose of RNA comprising 25 ug RNA encoding a SARS-CoV-2 S proteinfrom a BA.1 Omicron variant and 25 ug of RNA encoding a SARS-CoV-2 Sprotein having one or more mutations that are characteristic of a BA.4or BA.5 Omicron variant.

In some embodiments, a subject is administered (i) a primary regimencomprising at least two 30 ug doses of RNA encoding a SARS-CoV-2 Sprotein from a Wuhan strain, wherein the two doses are administered atleast approximately 21 days apart, and (ii) a booster regimen comprisinga 50 ug dose of RNA comprising 25 ug RNA encoding a SARS-CoV-2 S proteinfrom a BA.2 Omicron variant and 25 ug of RNA encoding a SARS-CoV-2 Sprotein having one or more mutations that are characteristic of a BA.4or BA.5 Omicron variant.

In some embodiments, a subject is administered a booster regimencomprising at least one 60 ug dose of RNA. In some embodiments, asubject is administered a booster regimen comprising 60 ug of RNAencoding a SARS-CoV-2 S protein from a Wuhan variant. In someembodiments, a subject is administered 60 ug of RNA encoding aSARS-CoV-2 S protein having one or more mutations that arecharacteristic of a SARS-CoV-2 variant (e.g., a variant describedherein). In some embodiments, a subject is administered a boosterregimen comprising 60 ug of RNA encoding a SARS-CoV-2 S protein havingone or more mutations that are characteristic of an Omicron variant(e.g., a BA.1, BA.2, BA.4, or BA.5 Omicron variant). In someembodiments, a subject is administered a booster regimen comprising 60ug of RNA, wherein the RNA comprises a first RNA encoding a SARS-CoV-2 Sprotein from a Wuhan strain, and at least one additional RNA encoding aSARS-CoV-2 S protein comprising mutations that are characteristic of aSARS-CoV-2 variant (e.g., in some embodiments, a subject is administereda booster regimen comprising 30 ug of RNA encoding a SARS-CoV-2 Sprotein from a Wuhan strain and 30 ug of RNA encoding a SARS-CoV-2 Sprotein having one or more mutations that are characteristic of anOmicron variant (e.g., a BA.1, BA.2, BA.4, or BA.5 variant).

In some embodiments, a subject is administered a booster regimencomprising at least one dose comprising 30 ug of RNA encoding aSARS-CoV-2 S protein from a Wuhan strain and 30 ug of RNA encoding aSARS-CoV-2 S protein having one or more mutations that arecharacteristic of a BA.1 Omicron variant. In some embodiments, a subjectis administered a booster regimen comprising at least one dosecomprising 20 ug of RNA encoding a SARS-CoV-2 S protein from a Wuhanstrain and 40 ug of RNA encoding a SARS-CoV-2 S protein having one ormore mutations that are characteristic of a BA.1 Omicron variant. Insome embodiments, a subject is administered a booster regimen comprisingat least one dose comprising 15 ug of RNA encoding a SARS-CoV-2 Sprotein from a Wuhan strain and 45 ug of RNA encoding a SARS-CoV-2 Sprotein having one or more mutations that are characteristic of a BA.1Omicron variant. In some embodiments, a subject is administered abooster regimen comprising at least one dose comprising 30 ug of RNAencoding a SARS-CoV-2 S protein from a Wuhan strain and 30 ug of RNAencoding a SARS-CoV-2 S protein having one or more mutations that arecharacteristic of a BA.2 Omicron variant. In some embodiments, a subjectis administered a booster regimen comprising at least one dosecomprising 20 ug of RNA encoding a SARS-CoV-2 S protein from a Wuhanstrain and 40 ug of RNA encoding a SARS-CoV-2 S protein having one ormore mutations that are characteristic of a BA.2 Omicron variant. Insome embodiments, a subject is administered a booster regimen comprisingat least one dose comprising 15 ug of RNA encoding a SARS-CoV-2 Sprotein from a Wuhan strain and 45 ug of RNA encoding a SARS-CoV-2 Sprotein having one or more mutations that are characteristic of a BA.2Omicron variant. In some embodiments, a subject is administered abooster regimen comprising at least one dose comprising 30 ug of RNAencoding a SARS-CoV-2 S protein from a Wuhan strain and 30 ug of RNAencoding a SARS-CoV-2 S protein having one or more mutations that arecharacteristic of a BA.3 Omicron variant. In some embodiments, a subjectis administered a booster regimen comprising at least one dosecomprising 20 ug of RNA encoding a SARS-CoV-2 S protein from a Wuhanstrain and 40 ug of RNA encoding a SARS-CoV-2 S protein having one ormore mutations that are characteristic of a BA.3 Omicron variant. Insome embodiments, a subject is administered a booster regimen comprisingat least one dose comprising 15 ug of RNA encoding a SARS-CoV-2 Sprotein from a Wuhan strain and 45 ug of RNA encoding a SARS-CoV-2 Sprotein having one or more mutations that are characteristic of a BA.3Omicron variant. In some embodiments, a subject is administered abooster regimen comprising at least one dose comprising 30 ug of RNAencoding a SARS-CoV-2 S protein from a Wuhan strain and 30 ug of RNAencoding a SARS-CoV-2 S protein having one or more mutations that arecharacteristic of a BA.4 or BA.5 Omicron variant. In some embodiments, asubject is administered a booster regimen comprising at least one dosecomprising 20 ug of RNA encoding a SARS-CoV-2 S protein from a Wuhanstrain and 40 ug of RNA encoding a SARS-CoV-2 S protein having one ormore mutations that are characteristic of a BA.4 or BA.5 Omicronvariant. In some embodiments, a subject is administered a boosterregimen comprising at least one dose comprising 15 ug of RNA encoding aSARS-CoV-2 S protein from a Wuhan strain and 45 ug of RNA encoding aSARS-CoV-2 S protein having one or more mutations that arecharacteristic of a BA.4 or BA.5 Omicron variant.

In some embodiments, a subject is administered a booster regimencomprising at least one dose comprising 30 ug of RNA encoding aSARS-CoV-2 S protein from a BA.1 Omicron variant and 30 ug of RNAencoding a SARS-CoV-2 S protein having one or more mutations that arecharacteristic of a BA.2 Omicron variant. In some embodiments, a subjectis administered a booster regimen comprising at least one dosecomprising 20 ug of RNA encoding a SARS-CoV-2 S protein from a BA.1Omicron variant and 40 ug of RNA encoding a SARS-CoV-2 S protein havingone or more mutations that are characteristic of a BA.2 Omicron variant.In some embodiments, a subject is administered a booster regimencomprising at least one dose comprising 15 ug of RNA encoding aSARS-CoV-2 S protein from a BA.1 Omicron variant and 45 ug of RNAencoding a SARS-CoV-2 S protein having one or more mutations that arecharacteristic of a BA.2 Omicron variant. In some embodiments, a subjectis administered a booster regimen comprising at least one dosecomprising 30 ug of RNA encoding a SARS-CoV-2 S protein from a BA.1Omicron variant and 30 ug of RNA encoding a SARS-CoV-2 S protein havingone or more mutations that are characteristic of a BA.3 Omicron variant.In some embodiments, a subject is administered a booster regimencomprising at least one dose comprising 20 ug of RNA encoding aSARS-CoV-2 S protein from a BA.1 Omicron variant and 40 ug of RNAencoding a SARS-CoV-2 S protein having one or more mutations that arecharacteristic of a BA.3 Omicron variant. In some embodiments, a subjectis administered a booster regimen comprising at least one dosecomprising 15 ug of RNA encoding a SARS-CoV-2 S protein from a BA.1Omicron variant and 45 ug of RNA encoding a SARS-CoV-2 S protein havingone or more mutations that are characteristic of a BA.3 Omicron variant.In some embodiments, a subject is administered a booster regimencomprising at least one dose comprising 30 ug of RNA encoding aSARS-CoV-2 S protein from a BA.1 Omicron variant and 30 ug of RNAencoding a SARS-CoV-2 S protein having one or more mutations that arecharacteristic of a BA.4 or BA.5 Omicron variant. In some embodiments, asubject is administered a booster regimen comprising at least one dosecomprising 20 ug of RNA encoding a SARS-CoV-2 S protein from a BA.1Omicron variant and 40 ug of RNA encoding a SARS-CoV-2 S protein havingone or more mutations that are characteristic of a BA.4 or BA.5 Omicronvariant. In some embodiments, a subject is administered a boosterregimen comprising at least one dose comprising 15 ug of RNA encoding aSARS-CoV-2 S protein from a BA.1 Omicron variant and 45 ug of RNAencoding a SARS-CoV-2 S protein having one or more mutations that arecharacteristic of a BA.4 or BA.5 Omicron variant.

In some embodiments, a subject is administered a booster regimencomprising at least one dose comprising 30 ug of RNA encoding aSARS-CoV-2 S protein from a BA.2 Omicron variant and 30 ug of RNAencoding a SARS-CoV-2 S protein having one or more mutations that arecharacteristic of a BA.4 or BA.5 Omicron variant. In some embodiments, asubject is administered a booster regimen comprising at least one dosecomprising 20 ug of RNA encoding a SARS-CoV-2 S protein from a BA.2Omicron variant and 40 ug of RNA encoding a SARS-CoV-2 S protein havingone or more mutations that are characteristic of a BA.4 or BA.5 Omicronvariant. In some embodiments, a subject is administered a boosterregimen comprising at least one dose comprising 15 ug of RNA encoding aSARS-CoV-2 S protein from a BA.2 Omicron variant and 45 ug of RNAencoding a SARS-CoV-2 S protein having one or more mutations that arecharacteristic of a BA.4 or BA.5 Omicron variant. In some embodiments, asubject is administered a booster regimen comprising at least one dosecomprising 30 ug of RNA encoding a SARS-CoV-2 S protein from a BA.3Omicron variant and 30 ug of RNA encoding a SARS-CoV-2 S protein havingone or more mutations that are characteristic of a BA.4 or BA.5 Omicronvariant. In some embodiments, a subject is administered a boosterregimen comprising at least one dose comprising 20 ug of RNA encoding aSARS-CoV-2 S protein from a BA.3 Omicron variant and 40 ug of RNAencoding a SARS-CoV-2 S protein having one or more mutations that arecharacteristic of a BA.4 or BA.5 Omicron variant. In some embodiments, asubject is administered a booster regimen comprising at least one dosecomprising 15 ug of RNA encoding a SARS-CoV-2 S protein from a BA.3Omicron variant and 45 ug of RNA encoding a SARS-CoV-2 S protein havingone or more mutations that are characteristic of a BA.4 or BA.5 Omicronvariant.

In some embodiments, a subject is administered (i) a primary regimencomprising two 30 ug doses of RNA encoding a SARS-CoV-2 S protein from aWuhan strain of SARS-CoV-2, wherein the two doses are administeredapproximately 21 days apart, and (ii) a booster regimen comprising atleast one 60 ug dose of RNA encoding a SARS-CoV-2 S protein from a Wuhanstrain of SARS-CoV-2, wherein the booster regimen is administered atleast two months (including, e.g., at least three months, at least fourmonths, at least five months, at least six months, or more) aftercompletion of the primary regimen.

In some embodiments, a subject is administered (i) a primary regimencomprising two 30 ug doses of RNA encoding a SARS-CoV-2 S protein from aWuhan strain, wherein the two doses are administered approximately 21days apart, and (ii) a booster regimen comprising at least one 60 ugdose of RNA encoding a SARS-CoV-2 S protein having one or mutations thatare characteristic of an Omicron variant of SARS-CoV-2 (e.g., a BA.1,BA.2, BA.4 or BA.5 Omicron variant), wherein the booster regimen isadministered at least two months (including, e.g., at least threemonths, at least four months, at least five months, at least six months,or more) after completion of the primary regimen. In some embodiments, asubject is administered (i) a primary regimen comprising two 30 ug dosesof RNA encoding a SARS-CoV-2 S protein from a Wuhan strain, wherein thetwo doses are administered approximately 21 days apart, and (iii) abooster regimen comprising at least one 60 ug dose of RNA comprising 30ug of RNA encoding a SARS-CoV-2 S protein having one or mutations thatare characteristic of an Omicron variant of SARS-CoV-2 and 30 ug of RNAencoding a SARS-CoV-2 S protein from a Wuhan strain, wherein a secondbooster regimen is administered at least two months (including, e.g., atleast three months, at least four months, at least five months, at leastsix months, or more) after completion of a first booster regimen.

In some embodiments, a subject is administered (i) a primary regimencomprising at least two 30 ug doses of RNA encoding a SARS-CoV-2 Sprotein from a Wuhan strain, wherein the two doses are administered atleast approximately 21 days apart, and (ii) a booster regimen comprisinga dose of 60 ug of RNA encoding a SARS-CoV-2 S protein having one ormutations that are characteristic of a BA.1 Omicron variant ofSARS-CoV-2, wherein the booster regimen is administered at least twomonths (including, e.g., at least three months, at least four months, atleast five months, at least six months, or more) after completion of theprimary regimen. In some embodiments, a subject is administered (i) aprimary regimen comprising at least two 30 ug doses of RNA encoding aSARS-CoV-2 S protein from a Wuhan strain, wherein the two doses areadministered at least approximately 21 days apart, and (ii) a boosterregimen comprising a 60 ug dose of RNA comprising 30 ug RNA encoding aSARS-CoV-2 S protein from a Wuhan strain and 30 ug of RNA encoding aSARS-CoV-2 S protein having one or more mutations that arecharacteristic of a BA.1 Omicron variant.

In some embodiments, a subject is administered (i) a primary regimencomprising at least two 30 ug doses of RNA encoding a SARS-CoV-2 Sprotein from a Wuhan strain, wherein the two doses are administered atleast approximately 21 days apart, and (ii) a booster regimen comprisinga dose of 60 ug of RNA encoding a SARS-CoV-2 S protein having one ormutations that are characteristic of a BA.2 Omicron variant ofSARS-CoV-2, wherein the booster regimen is administered at least twomonths (including, e.g., at least three months, at least four months, atleast five months, at least six months, or more) after completion of theprimary regimen. In some embodiments, a subject is administered (i) aprimary regimen comprising at least two 30 ug doses of RNA encoding aSARS-CoV-2 S protein from a Wuhan strain, wherein the two doses areadministered at least approximately 21 days apart, and (ii) a boosterregimen comprising a 60 ug dose of RNA comprising 30 ug RNA encoding aSARS-CoV-2 S protein from a Wuhan strain and 30 ug of RNA encoding aSARS-CoV-2 S protein having one or more mutations that arecharacteristic of a BA.2 Omicron variant.

In some embodiments, a subject is administered (i) a primary regimencomprising at least two 30 ug doses of RNA encoding a SARS-CoV-2 Sprotein from a Wuhan strain, wherein the two doses are administered atleast approximately 21 days apart, and (ii) a booster regimen comprisinga dose of 60 ug of RNA encoding a SARS-CoV-2 S protein having one ormutations that are characteristic of a BA.4 or BA.5 Omicron variant ofSARS-CoV-2, wherein the booster regimen is administered at least twomonths (including, e.g., at least three months, at least four months, atleast five months, at least six months, or more) after completion of theprimary regimen. In some embodiments, a subject is administered (i) aprimary regimen comprising at least two 30 ug doses of RNA encoding aSARS-CoV-2 S protein from a Wuhan strain, wherein the two doses areadministered at least approximately 21 days apart, and (ii) a boosterregimen comprising a 60 ug dose of RNA comprising 30 ug RNA encoding aSARS-CoV-2 S protein from a Wuhan strain and 30 ug of RNA encoding aSARS-CoV-2 S protein having one or more mutations that arecharacteristic of a BA.4/5 Omicron variant.

In some embodiments, a subject is administered (i) a primary regimencomprising at least two 30 ug doses of RNA encoding a SARS-CoV-2 Sprotein from a Wuhan strain, wherein the two doses are administered atleast approximately 21 days apart, and (ii) a booster regimen comprisinga 60 ug dose of RNA comprising 30 ug RNA encoding a SARS-CoV-2 S proteinfrom a BA.1 Omicron variant and 30 ug of RNA encoding a SARS-CoV-2 Sprotein having one or more mutations that are characteristic of a BA.2Omicron variant.

In some embodiments, a subject is administered (i) a primary regimencomprising at least two 30 ug doses of RNA encoding a SARS-CoV-2 Sprotein from a Wuhan strain, wherein the two doses are administered atleast approximately 21 days apart, and (ii) a booster regimen comprisinga 60 ug dose of RNA comprising 30 ug RNA encoding a SARS-CoV-2 S proteinfrom a BA.1 Omicron variant and 30 ug of RNA encoding a SARS-CoV-2 Sprotein having one or more mutations that are characteristic of a BA.4or BA.5 Omicron variant.

In some embodiments, a subject is administered (i) a primary regimencomprising at least two 30 ug doses of RNA encoding a SARS-CoV-2 Sprotein from a Wuhan strain, wherein the two doses are administered atleast approximately 21 days apart, and (ii) a booster regimen comprisinga 60 ug dose of RNA comprising 30 ug RNA encoding a SARS-CoV-2 S proteinfrom a BA.2 Omicron variant and 30 ug of RNA encoding a SARS-CoV-2 Sprotein having one or more mutations that are characteristic of a BA.4or BA.5 Omicron variant.

In some embodiments, a patient is administered a primary regimencomprising two 30 ug doses, administered approximately 21 days apart,and a booster regimen comprising at least one 60 ug dose of RNA (e.g.,60 ug of RNA encoding a SARS-CoV-2 S protein from a Wuhan strain, 60 ugof RNA encoding a SARS-CoV-2 S protein having one or more mutations thatare characteristic of an Omicron variant (e.g., a BA.1, BA.2, BA.4, orBA.5 Omicron variant), or 30 ug of RNA encoding a SARS-CoV-2 S proteinfrom a Wuhan strain and 30 ug of RNA encoding a SARS-CoV-2 S proteinhaving one or more mutations that are characteristic of an Omicronvariant). In some embodiments, a patient is administered a primaryregimen comprising two 30 ug doses, administered approximately 21 daysapart, and a booster regimen comprising at least one 50 ug dose of RNA(e.g., 50 ug of RNA encoding a SARS-CoV-2 S protein from a Wuhan strain,50 ug of RNA encoding a SARS-CoV-2 S protein having one or moremutations that are characteristic of an Omicron variant, or 25 ug of RNAencoding a SARS-CoV-2 S protein from a Wuhan strain and 25 ug of RNAencoding a SARS-CoV-2 S protein having one or more mutations that arecharacteristic of an Omicron variant). In some embodiments, a patient isadministered a primary regimen comprising two 30 ug doses, administeredapproximately 21 days apart, and a booster regimen comprising at leastone 30 ug dose of RNA (e.g., 30 ug of RNA encoding a SARS-CoV-2 Sprotein from a Wuhan strain, 30 ug of RNA encoding a SARS-CoV-2 Sprotein having one or more mutations that are characteristic of anOmicron variant, or 15 ug of RNA encoding a SARS-CoV-2 S protein from aWuhan strain and 15 ug of RNA encoding a SARS-CoV-2 S protein having oneor more mutations that are characteristic of an Omicron variant).

In some embodiments, a primary regimen may involve one or more 30 ugdoses and a booster regimen may involve one or more 20 ug doses, one ormore 10 ug doses, and/or one or more 3 ug doses. In some embodiments, aprimary regimen may involve one or more 20 ug doses and a boosterregimen may involve one or more 10 ug doses, and/or one or more 3 ugdoses. In some embodiments, a primary regimen may involve one or more 10ug doses and a booster regimen may involve one or more 3 ug doses. Insome embodiments, a primary regimen may involve one or more 3 ug doses,and a booster regimen may also involve one or more 3 ug doses.

In some embodiments, a booster regimen comprises a single dose, e.g.,for patients who experienced an adverse reaction while receiving theprimary regimen.

In some embodiments, the same RNA as used in a primary regimen is usedin a booster regimen. In some embodiment, an RNA used in primary andbooster regimens is BNT162b2. In some embodiments, a different RNA isused in a booster regimen relative to that used in a primary regimenadministered to the same subject. In some embodiments, BNT162b2 is usedin a primary regimen but not in a booster regimen. In some embodiments,BNT162b2 is used in a booster regimen but not in a primary regimen. Insome embodiments, a similar BNT162b2 construct can be used in a primaryregimen and in a booster regimen, except that the RNA constructs used inthe primary and booster regimens encode a SARS-CoV-2 S protein (or animmunogenic portion thereof) of different SARS-CoV-2 strains (e.g., asdescribed herein).

In some embodiments, where BNT162b2 is used for a primary regimen or abooster regimen but not both, and a different RNA is used in the other,such different RNA may be an RNA encoding the same SARS-CoV-2 S proteinbut with different codon optimization or other different RNA sequence.In some embodiments, such different RNA may encode a SARS-CoV-2 Sprotein (or an immunogenic portion thereof) of a different SARS-CoV-2strain, e.g., of a variant strain discussed herein. In some suchembodiments, such variant strain that is prevalent or rapidly spreadingin a relevant jurisdiction. In some embodiments, such different RNA maybe an RNA encoding a SARS-CoV-2 S protein or variant thereof (orimmunogenic portion of either) comprising one or more mutationsdescribed herein for S protein variants such as SARS-CoV-2 S proteinvariants, in particular naturally occurring S protein variants; in somesuch embodiments, a SARS-CoV-2 variant may be selected from the groupconsisting of VOC-202012/01, 501.V2, Cluster 5 and B.1.1.248. In someembodiments, a SARS-CoV-2 variant may be selected from the groupconsisting of VOC-202012/01, 501.V2, Cluster 5 and B.1.1.248, B.1.1.7,B.1.617.2, and B.1.1.529. In some embodiments, a booster regimencomprises at least one dose of RNA that encodes a SARS-CoV-2 S protein(or an immunogenic fragment thereof) of a variant that is spreadingrapidly in a relevant jurisdiction at the time of administration. Insome such embodiments, a variant that is encoded by RNA administered ina booster regimen may be different from that encoded by RNA administeredin a primary regimen.

In some embodiments, a booster regimen comprises administering (i) adose of RNA encoding the same SARS-CoV-2 S protein (or an immunogenicfragment thereof) as the RNA administered in the primary regimen (e.g.,an RNA encoding a SARS-CoV-2 the S protein (or an immunogenic fragmentthereof) from the SARS-CoV-2 Wuhan strain) and (ii) a dose of RNAencoding a SARS-CoV-2 S protein (or an immunogenic fragment thereof) ofa variant that is spreading rapidly in a relevant jurisdiction at thetime of administration (e.g., a SARS-CoV-2 S protein (or an immunogenicfragment thereof) from one of the SARS-CoV-2 variants discussed herein).

In some embodiments, a booster regimen comprises multiple doses (e.g.,at least two doses, at least three doses, or more). For example, in someembodiments, a first dose of a booster regimen may comprise an RNAencoding the same SARS-CoV-2 S protein (or an immunogenic fragmentthereof) administered in the primary regimen and a second dose of abooster regimen may comprise the RNA encoding a SARS-CoV-2 S protein ofa variant that is spreading rapidly in a relevant jurisdiction at thetime of administration. In some embodiments, a first dose of a boosterregimen may comprise RNA encoding a SARS-CoV-2 S protein (or animmunogenic fragment thereof) of a variant that is spreading rapidly ina relevant jurisdiction at the time of administration and a second doseof a booster regimen may comprise RNA encoding the same SARS-CoV-2 Sprotein (or an immunogenic fragment thereof) administered in the primaryregimen. In some embodiments, the booster regimen comprises multipledoses, and the RNA encoding the S protein of a variant that is spreadingrapidly in a relevant jurisdiction is administered in a first dose andthe RNA encoding the S protein administered in the primary regimen isadministered in a second dose.

In some embodiments, doses (e.g., a first and a second dose or any twoconsecutive doses) in a booster regimen are administered at least 2weeks apart, including, e.g., at least 3 weeks, at least 4 weeks, atleast 5 weeks, at least 6 weeks, at least 7 week, at least 8 weeks, atleast 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks,at least 13 weeks, at least 14 weeks, at least 15 weeks, at least 16weeks, or longer, apart. In some embodiments, doses (e.g., a first and asecond dose or any two consecutive doses) in a booster regimen areadministered approximately 2 to 168 weeks apart. In some embodiments,doses (e.g., a first and a second dose or any two consecutive doses) ina booster regimen are administered approximately 3 to 12 weeks apart. Insome embodiments, doses (e.g., a first and a second dose or any twoconsecutive doses) in a booster regimen are administered approximately 4to 10 weeks apart. In some embodiments, doses (e.g., a first and asecond dose or any two consecutive doses) in a booster regimen areadministered approximately 6 to 8 weeks apart. (e.g., about 21 daysapart, or about 6 to 8 weeks apart). In some embodiments, the first andsecond dose are administered on the same day (e.g., by intramuscularinjection at different sites on the subject).

In such embodiments, the booster regimen can optionally further comprisea third and fourth dose, administered approximately 2 to 8 weeks afterthe first and second dose (e.g., about 21 days after the first andsecond dose, or about 6 weeks to about 8 weeks after the first andsecond dose), where the third and fourth dose are also administered onthe same day (e.g., by intramuscular injection at different sites on thesubject), and comprise the same RNAs administered in the first andsecond doses of the booster regimen.

In some embodiments, multiple booster regimens may be administered. Insome embodiments, a booster regimen is administered to a patient who haspreviously been administered a booster regimen.

In some embodiments, a second booster regimen is administered to apatient who has previously received a first booster regimen, and theamount of RNA administered in at least one dose of a second boosterregimen is higher than the amount of RNA administered in at least onedose of a first booster regimen.

In some embodiments, a second booster regimen comprises administering atleast one dose of 3 ug of RNA. In some embodiments, a second boosterregimen comprises administering at least one dose of 5 ug of RNA. Insome embodiments, a second booster regimen comprises administering atleast one dose of 10 ug of RNA. In some embodiments, a second boosterregimen comprises administering at least one dose of 15 ug of RNA. Insome embodiments, a second booster regimen comprises administering atleast one dose of 20 ug of RNA. In some embodiments, a second boosterregimen comprises administering at least one dose of 25 ug of RNA. Insome embodiments, a second booster regimen comprises administering atleast one dose of 30 ug of RNA. In some embodiments, a second boosterregimen comprises administering at least one dose of 50 ug of RNA. Insome embodiments, a second booster regimen comprises administering atleast one dose of 60 ug of RNA.

In some embodiments, a subject is administered a primary regimen thatcomprises two doses of 30 ug of RNA, administered approximately 21 daysapart, and a booster regimen comprising at least one dose ofapproximately 30 ug of RNA. In some embodiments, a subject isadministered a primary regimen that comprises two doses of 30 ug of RNA,administered approximately 21 days apart, and a booster regimencomprising at least one dose of approximately 50 ug of RNA. In someembodiments, a subject is administered a primary regimen that comprisestwo doses of 30 ug of RNA, administered approximately 21 days apart, anda booster regimen comprising at least one dose of approximately 60 ug ofRNA.

In some embodiments, a subject is administered a primary regimen thatcomprises two doses of 30 ug of RNA, administered approximately 21 daysapart, a first booster regimen comprising at least one dose ofapproximately 30 ug of RNA, and a second booster regimen comprising atleast one dose of approximately 30 ug of RNA. In some embodiments, asubject is administered a primary regimen that comprises two doses of 30ug of RNA, administered approximately 21 days apart, a first boosterregimen comprising at least one dose of approximately 30 ug of RNA, anda second booster regimen comprising at least one dose of approximately50 ug of RNA. In some embodiments, a subject is administered a primaryregimen that comprises two doses of 30 ug of RNA, administeredapproximately 21 days apart, a first booster regimen comprising at leastone dose of approximately 30 ug of RNA, and a second booster regimencomprising at least one dose of approximately 60 ug of RNA. In someembodiments, a first booster regimen comprises two doses of RNA, whereineach dose comprises an RNA encoding a Spike protein from a differentSARS-CoV-2 variant. In some embodiments, a first booster regimencomprises two doses of RNA, wherein each dose comprises an RNA encodinga Spike protein from a different SARS-CoV-2 variant, and wherein the twodoses of RNA are administered on the same day. In some embodiments, thetwo doses of RNA are administered in a single composition (e.g., bymixing a first composition comprising an RNA encoding a Spike proteinfrom a first SARS-CoV-2 variant with a second composition comprising anRNA encoding a Spike protein from a second SARS-CoV-2 variant).

In some embodiments, a subject is administered a booster regimencomprising a first dose comprising an RNA that encodes a Spike proteinfrom a Wuhan strain of SARS-CoV-2 and a second dose comprising an RNAthat encodes a Spike protein comprising mutations from a variant that isprevalent and/or rapidly spreading in a relevant jurisdiction at thetime of administering the booster regimen, wherein the first dose andthe second dose of RNA may be administered on the same day. In someembodiments, a subject is administered a booster regimen comprising afirst dose comprising an RNA that encodes a Spike protein from a Wuhanstrain of SARS-CoV-2 and a second dose comprising an RNA that encodes aSpike protein comprising mutations from an alpha variant of SARS-CoV-2,wherein the first dose and the second dose may be administered on thesame day. In some embodiments, a subject is administered a boosterregimen comprising a first dose comprising an RNA that encodes a Spikeprotein from a Wuhan strain of SARS-CoV-2 and a second dose comprisingan RNA that encodes a Spike protein comprising mutations from a betavariant of SARS-CoV-2, wherein the first dose and the second dose may beadministered on the same day. In some embodiments, a subject isadministered a booster regimen comprising a first dose comprising an RNAthat encodes a Spike protein from a Wuhan strain of SARS-CoV-2 and asecond dose comprising an RNA that encodes a Spike protein comprisingmutations from a delta variant of SARS-CoV-2, wherein the first dose andthe second dose may be administered on the same day. In someembodiments, a subject is administered a booster regimen comprising afirst dose comprising an RNA that encodes a Spike protein from a Wuhanstrain of SARS-CoV-2 and a second dose comprising an RNA that encodes aSpike protein comprising mutations from an Omicron variant ofSARS-CoV-2, wherein the first dose and the second dose may beadministered on the same day. Such booster regimens may be administered,e.g., to a subject previously administered a primary dosing regimenand/or to a subject previously administered a primary dosing regimen anda booster regimen.

In some embodiments, a subject is administered a first booster regimencomprising a first dose of 15 ug of RNA encoding a Spike protein from aWuhan variant and a second dose of 15 ug of RNA encoding a Spike proteinfrom an Omicron variant of SARS-CoV-2, where the first and the seconddose are administered on the same day (e.g., wherein compositionscomprising the RNA are mixed prior to administration, and the mixture isthen administered to a patient). In some embodiments, a subject isadministered a first booster regimen comprising a first dose of 25 ug ofRNA encoding a Spike protein from a Wuhan variant and a second dose of25 ug of RNA encoding a Spike protein from an Omicron variant ofSARS-CoV-2. In some embodiments, the first and the second doses areoptionally administered on the same day. In some embodiments, a subjectis administered a first booster regimen comprising a first dose of 25 ugof RNA encoding a Spike protein from a Wuhan variant and a second doseof 25 ug of RNA encoding a Spike protein from an Omicron variant ofSARS-CoV-2. In some embodiments, the first and the second doses areadministered on the same day. In some embodiments, a subject isadministered a first booster regimen comprising a first dose of 30 ug ofRNA encoding a Spike protein from a Wuhan variant and a second dose of30 ug of RNA encoding a Spike protein from an Omicron variant ofSARS-CoV-2, wherein the first and the second dose are optionallyadministered on the same day (e.g., in separate administrations or asadministration of a multivalent vaccine). In some embodiments, such afirst booster regimen is administered to a subject previouslyadministered a primary regimen comprising two doses of 30 ug of RNA,administered about 21 days apart wherein the first booster regimen isadministered at least 3 months (e.g., at least 4, at least 5, or atleast 6 months) after administration of a primary regimen.

In some embodiments, a subject is administered a second booster regimencomprising a first dose of 15 ug of RNA encoding a Spike protein from aWuhan variant and a second dose of 15 ug of RNA encoding a Spike proteinfrom an Omicron variant of SARS-CoV-2, where the first and the seconddose are administered on the same day (e.g., wherein compositionscomprising the RNA are mixed prior to administration to form amultivalent vaccine, and the mixture is then administered to a patient).In some embodiments, a subject is administered a second booster regimencomprising a first dose of 25 ug of RNA encoding a Spike protein from aWuhan variant and a second dose of 25 ug of RNA encoding a Spike proteinfrom an Omicron variant of SARS-CoV-2, wherein the first dose and thesecond dose are optionally administered on the same day (e.g., viaadministration of a multivalent vaccine or via administration ofseparate compositions). In some embodiments, a subject is administered asecond booster regimen comprising a first dose of 25 ug of RNA encodinga Spike protein from a Wuhan variant and a second dose of 25 ug of RNAencoding a Spike protein from an Omicron variant of SARS-CoV-2. In someembodiments, a subject is administered a second booster regimencomprising a first dose of 30 ug of RNA encoding a Spike protein from aWuhan variant and a second dose of 30 ug of RNA encoding a Spike proteinfrom an Omicron variant of SARS-CoV-2, wherein the first dose and thesecond dose are optionally administered on the same day (e.g., viaadministration of a multivalent vaccine or via administration ofseparate compositions). In some embodiments, such a second boosterregimen is administered to a subject previously administered a primaryregimen comprising two doses of 30 ug of RNA, administered about 21 daysapart. In some embodiments, such a second booster regimen isadministered to a subject previously administered a primary regimencomprising two doses of 30 ug of RNA, administered about 21 days apart,and a first booster regimen comprising a dose of 30 ug of RNA, whereinthe second booster regimen is administered at least 3 months (e.g., atleast 4, at least 5, or at least 6 months) after administration of afirst booster regimen.

In some embodiments, patients receiving dose(s) of RNA compositions asdescribed herein are monitored for one or more particular conditions,e.g., following administration of one or more doses. In someembodiments, such condition(s) may be or comprise allergic reaction(s)(particularly in subject(s) with a history of relevant allergies orallergic reactions), myocarditis (inflammation of the heart muscle,particularly where the subject is a young male and/or may haveexperienced prior such inflammation), pericarditis (inflammation of thelining outside the heart, particularly where the subject is a youngmales and/or may have experienced prior such inflammation), fever,bleeding (particularly where the subject is known to have a bleedingdisorder or to be receiving therapy with a blood thinner). Alternativelyor additionally, patients who may receive closer monitoring may be orinclude patients who are immunocompromised or are receiving therapy witha medicine that affects the immune system, are pregnant or planning tobecome pregnant, are breastfeeding, have received another COVID-19vaccine, and/or have ever fainted in association with an injection. Insome embodiments, patients are monitored for myocarditis followingadministration of one of the compositions disclosed herein. In someembodiments, patients are monitored for pericarditis followingadministration of one of the compositions disclosed herein. Patients maybe monitored and/or treated for the condition using current standards ofcare.

In some embodiments, efficacy for RNA (e.g., mRNA) compositionsdescribed in pediatric populations (e.g., described herein) may beassessed by various metrics described herein (including, e.g., but notlimited to COVID-19 incidence per 1000 person-years in subjects with noserological or virological evidence of past SARS-CoV-2 infection;geometric mean ratio (GMR) of SARS CoV-2 neutralizing titers measured,e.g., 7 days after a second dose; etc.) In some embodiments, pediatricpopulations described herein (e.g., from 12 to less than 16 years ofage) may be monitored for occurrence of multisystem inflammatorysyndrome (MIS) (e.g., inflammation in different body parts such as,e.g., heart, lung, kidneys, brain, skin, eyes, and/or gastrointestinalorgans), after administration of an RNA composition (e.g., mRNA)described herein. Exemplary symptoms of MIS in children may include, butare not limited to fever, abdominal pain, vomiting, diarrhea, neck pain,rash, bloodshot eyes, feeling extra tried, and combinations thereof.

In one embodiment, RNA administered as described above is nucleosidemodified messenger RNA (modRNA) described herein as BNT162b1 (RBP020.3),BNT162b2 (RBP020.1 or RBP020.2), or BNT162b3 (e.g., BNT162b3c). In oneembodiment, RNA administered as described above is nucleoside modifiedmessenger RNA (modRNA) described herein as RBP020.2. In one embodiment,RNA encoding a vaccine antigen is nucleoside modified messenger RNA(modRNA) described herein as BNT162b3 (e.g., BNT162b3c).

In one embodiment, RNA administered as described above is nucleosidemodified messenger RNA (modRNA) and (i) comprises the nucleotidesequence of SEQ ID NO: 21, a nucleotide sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequenceof SEQ ID NO: 21, and/or (ii) encodes an amino acid sequence comprisingthe amino acid sequence of SEQ ID NO: 5, or an amino acid sequencehaving at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity tothe amino acid sequence of SEQ ID NO: 5. In one embodiment, RNAadministered as described above is nucleoside modified messenger RNA(modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 21;and/or (ii) encodes an amino acid sequence comprising the amino acidsequence of SEQ ID NO: 5.

In one embodiment, RNA administered as described above is nucleosidemodified messenger RNA (modRNA) and (i) comprises the nucleotidesequence of SEQ ID NO: 19, or 20, a nucleotide sequence having at least99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotidesequence of SEQ ID NO: 19, or 20, and/or (ii) encodes an amino acidsequence comprising the amino acid sequence of SEQ ID NO: 7, or an aminoacid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%identity to the amino acid sequence of SEQ ID NO: 7. In one embodiment,RNA administered as described above is nucleoside modified messenger RNA(modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 19, or20; and/or (ii) encodes an amino acid sequence comprising the amino acidsequence of SEQ ID NO: 7.

In one embodiment, RNA administered as described above is nucleosidemodified messenger RNA (modRNA) and (i) comprises the nucleotidesequence of SEQ ID NO: 20, a nucleotide sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequenceof SEQ ID NO: 20, and/or (ii) encodes an amino acid sequence comprisingthe amino acid sequence of SEQ ID NO: 7, or an amino acid sequencehaving at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity tothe amino acid sequence of SEQ ID NO: 7. In one embodiment, RNAadministered as described above is nucleoside modified messenger RNA(modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 20;and/or (ii) encodes an amino acid sequence comprising the amino acidsequence of SEQ ID NO: 7.

In one embodiment, RNA administered as described above is nucleosidemodified messenger RNA (modRNA) and (i) comprises the nucleotidesequence of SEQ ID NO: 30, a nucleotide sequence having at least 99%,98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequenceof SEQ ID NO: 30, and/or (ii) encodes an amino acid sequence comprisingthe amino acid sequence of SEQ ID NO: 29, or an amino acid sequencehaving at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity tothe amino acid sequence of SEQ ID NO: 29. In one embodiment, RNAadministered as described above is nucleoside modified messenger RNA(modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 30;and/or (ii) encodes an amino acid sequence comprising the amino acidsequence of SEQ ID NO: 29.

In one embodiment, RNA administered is nucleoside modified messenger RNA(modRNA), (i) comprises the nucleotide sequence of SEQ ID NO: 20; and/or(ii) encodes an amino acid sequence comprising the amino acid sequenceof SEQ ID NO: 7, and is administered in an amount of about 30 μg perdose. In one embodiment, at least two of such doses are administered.For example, a second dose may be administered about 21 days followingadministration of the first dose.

In some embodiments, populations to be treated with RNA described hereincomprise, essentially consist of, or consist of subjects of age of atleast 50, at least 55, at least 60, or at least 65. In some embodiments,populations to be treated with RNA described herein comprise,essentially consist of, or consist of subjects of age of between 55 to90, 60 to 85, or 65 to 85.

In some embodiments, the period of time between the doses administeredis at least 7 days, at least 14 days, or at least 21 days. In someembodiments, the period of time between the doses administered isbetween 7 days and 28 days such as between 14 days and 23 days.

In some embodiments, no more than 5 doses, no more than 4 doses, or nomore than 3 doses of the RNA described herein may be administered to asubject.

In some embodiments, the methods and agents described herein areadministered (in a regimen, e.g., at a dose, frequency of doses and/ornumber of doses) such that adverse events (AE), i.e., any unwantedmedical occurrence in a patient, e.g., any unfavourable and unintendedsign, symptom, or disease associated with the use of a medicinalproduct, whether or not related to the medicinal product, are mild ormoderate in intensity. In some embodiments, the methods and agentsdescribed herein are administered such that adverse events (AE) can bemanaged with interventions such as treatment with, e.g., paracetamol orother drugs that provide analgesic, antipyretic (fever-reducing) and/oranti-inflammatory effects, e.g., nonsteroidal anti-inflammatory drugs(NSAIDs), e.g., aspirin, ibuprofen, and naproxen. Paracetamol or“acetaminophen” which is not classified as a NSAID exerts weakanti-inflammatory effects and can be administered as analgesic accordingto the present disclosure.

In some embodiments, the methods and agents described herein provide aneutralizing effect in a subject to coronavirus, coronavirus infection,or to a disease or disorder associated with coronavirus.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an immune response that blocks orneutralizes coronavirus in the subject. In some embodiments, the methodsand agents described herein following administration to a subject inducethe generation of antibodies such as IgG antibodies that block orneutralize coronavirus in the subject. In some embodiments, the methodsand agents described herein following administration to a subject inducean immune response that blocks or neutralizes coronavirus S proteinbinding to ACE2 in the subject. In some embodiments, the methods andagents described herein following administration to a subject induce thegeneration of antibodies that block or neutralize coronavirus S proteinbinding to ACE2 in the subject.

In some embodiments, the methods and agents described herein followingadministration to a subject induce geometric mean concentrations (GMCs)of RBD domain-binding antibodies such as IgG antibodies of at least 500U/ml, 1000 U/ml, 2000 U/ml, 3000 U/ml, 4000 U/ml, 5000 U/ml, 10000 U/ml,15000 U/ml, 20000 U/ml, 25000 U/ml, 30000 U/ml or even higher. In someembodiments, the elevated GMCs of RBD domain-binding antibodies persistfor at least 14 days, 21 days, 28 days, 1 month, 3 months, 6 months, 12months or even longer.

In some embodiments, the methods and agents described herein followingadministration to a subject induce geometric mean titers (GMTs) ofneutralizing antibodies such as IgG antibodies of at least 100 U/ml, 200U/ml, 300 U/ml, 400 U/ml, 500 U/ml, 1000 U/ml, 1500 U/ml, or evenhigher. In some embodiments, the elevated GMTs of neutralizingantibodies persist for at least 14 days, 21 days, 28 days, 1 month, 3months, 6 months, 12 months or even longer.

As used herein, the term “neutralization” refers to an event in whichbinding agents such as antibodies bind to a biological active site of avirus such as a receptor binding protein, thereby inhibiting the viralinfection of cells. As used herein, the term “neutralization” withrespect to coronavirus, in particular coronavirus S protein, refers toan event in which binding agents such as antibodies bind to the RBDdomain of the S protein, thereby inhibiting the viral infection ofcells. In particular, the term “neutralization” refers to an event inwhich binding agents eliminate or significantly reduce virulence (e.g.ability of infecting cells) of viruses of interest.

The type of immune response generated in response to an antigenicchallenge can generally be distinguished by the subset of T helper (Th)cells involved in the response. Immune responses can be broadly dividedinto two types: Th1 and Th2. Th1 immune activation is optimized forintracellular infections such as viruses, whereas Th2 immune responsesare optimized for humoral (antibody) responses. Th1 cells produceinterleukin 2 (IL-2), tumor necrosis factor (TNFα) and interferon gamma(IFNγ). Th2 cells produce IL-4, IL-5, IL-6, IL-9, IL-10 and IL-13. Th1immune activation is the most highly desired in many clinicalsituations. Vaccine compositions specialized in eliciting Th2 or humoralimmune responses are generally not effective against most viraldiseases.

In some embodiments, the methods and agents described herein followingadministration to a subject induce or promote a Th1-mediated immuneresponse in the subject. In some embodiments, the methods and agentsdescribed herein following administration to a subject induce or promotea cytokine profile that is typical for a Th1-mediated immune response inthe subject. In some embodiments, the methods and agents describedherein following administration to a subject induce or promote theproduction of interleukin 2 (IL-2), tumor necrosis factor (TNFα) and/orinterferon gamma (IFNγ) in the subject. In some embodiments, the methodsand agents described herein following administration to a subject induceor promote the production of interleukin 2 (IL-2) and interferon gamma(IFNγ) in the subject. In some embodiments, the methods and agentsdescribed herein following administration to a subject do not induce orpromote a Th2-mediated immune response in the subject, or induce orpromote a Th2-mediated immune response in the subject to a significantlower extent compared to the induction or promotion of a Th1-mediatedimmune response. In some embodiments, the methods and agents describedherein following administration to a subject do not induce or promote acytokine profile that is typical for a Th2-mediated immune response inthe subject, or induce or promote a cytokine profile that is typical fora Th2-mediated immune response in the subject to a significant lowerextent compared to the induction or promotion of a cytokine profile thatis typical for a Th1-mediated immune response. In some embodiments, themethods and agents described herein following administration to asubject do not induce or promote the production of IL-4, IL-5, IL-6,IL-9, IL-10 and/or IL-13, or induce or promote the production of IL-4,IL-5, IL-6, IL-9, IL-10 and/or IL-13 in the subject to a significantlower extent compared to the induction or promotion of interleukin 2(IL-2), tumor necrosis factor (TNFα) and/or interferon gamma (IFNγ) inthe subject. In some embodiments, the methods and agents describedherein following administration to a subject do not induce or promotethe production of IL-4, or induce or promote the production of IL-4 inthe subject to a significant lower extent compared to the induction orpromotion of interleukin 2 (IL-2) and interferon gamma (IFNγ) in thesubject.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a panel ofdifferent S protein variants such as SARS-CoV-2 S protein variants, inparticular naturally occurring S protein variants. In some embodiments,the panel of different S protein variants comprises at least 5, at least10, at least 15, or even more S protein variants. In some embodiments,such S protein variants comprise variants having amino acidmodifications in the RBD domain and/or variants having amino acidmodifications outside the RBD domain. In one embodiment, such S proteinvariant comprises SARS-CoV-2 S protein or a naturally occurring variantthereof wherein the amino acid corresponding to position 321 (Q) in SEQID NO: 1 is S. In one embodiment, such S protein variant comprisesSARS-CoV-2 S protein or a naturally occurring variant thereof whereinthe amino acid corresponding to position 321 (Q) in SEQ ID NO: 1 is L.In one embodiment, such S protein variant comprises SARS-CoV-2 S proteinor a naturally occurring variant thereof wherein the amino acidcorresponding to position 341 (V) in SEQ ID NO: 1 is I. In oneembodiment, such S protein variant comprises SARS-CoV-2 S protein or anaturally occurring variant thereof wherein the amino acid correspondingto position 348 (A) in SEQ ID NO: 1 is T. In one embodiment, such Sprotein variant comprises SARS-CoV-2 S protein or a naturally occurringvariant thereof wherein the amino acid corresponding to position 354 (N)in SEQ ID NO: 1 is D. In one embodiment, such S protein variantcomprises SARS-CoV-2 S protein or a naturally occurring variant thereofwherein the amino acid corresponding to position 359 (S) in SEQ ID NO: 1is N. In one embodiment, such S protein variant comprises SARS-CoV-2 Sprotein or a naturally occurring variant thereof wherein the amino acidcorresponding to position 367 (V) in SEQ ID NO: 1 is F. In oneembodiment, such S protein variant comprises SARS-CoV-2 S protein or anaturally occurring variant thereof wherein the amino acid correspondingto position 378 (K) in SEQ ID NO: 1 is S. In one embodiment, such Sprotein variant comprises SARS-CoV-2 S protein or a naturally occurringvariant thereof wherein the amino acid corresponding to position 378 (K)in SEQ ID NO: 1 is R. In one embodiment, such S protein variantcomprises SARS-CoV-2 S protein or a naturally occurring variant thereofwherein the amino acid corresponding to position 408 (R) in SEQ ID NO: 1is I. In one embodiment, such S protein variant comprises SARS-CoV-2 Sprotein or a naturally occurring variant thereof wherein the amino acidcorresponding to position 409 (Q) in SEQ ID NO: 1 is E. In oneembodiment, such S protein variant comprises SARS-CoV-2 S protein or anaturally occurring variant thereof wherein the amino acid correspondingto position 435 (A) in SEQ ID NO: 1 is S. In one embodiment, such Sprotein variant comprises SARS-CoV-2 S protein or a naturally occurringvariant thereof wherein the amino acid corresponding to position 439 (N)in SEQ ID NO: 1 is K. In one embodiment, such S protein variantcomprises SARS-CoV-2 S protein or a naturally occurring variant thereofwherein the amino acid corresponding to position 458 (K) in SEQ ID NO: 1is R. In one embodiment, such S protein variant comprises SARS-CoV-2 Sprotein or a naturally occurring variant thereof wherein the amino acidcorresponding to position 472 (1) in SEQ ID NO: 1 is V. In oneembodiment, such S protein variant comprises SARS-CoV-2 S protein or anaturally occurring variant thereof wherein the amino acid correspondingto position 476 (G) in SEQ ID NO: 1 is S. In one embodiment, such Sprotein variant comprises SARS-CoV-2 S protein or a naturally occurringvariant thereof wherein the amino acid corresponding to position 477 (S)in SEQ ID NO: 1 is N. In one embodiment, such S protein variantcomprises SARS-CoV-2 S protein or a naturally occurring variant thereofwherein the amino acid corresponding to position 483 (V) in SEQ ID NO: 1is A. In one embodiment, such S protein variant comprises SARS-CoV-2 Sprotein or a naturally occurring variant thereof wherein the amino acidcorresponding to position 508 (Y) in SEQ ID NO: 1 is H. In oneembodiment, such S protein variant comprises SARS-CoV-2 S protein or anaturally occurring variant thereof wherein the amino acid correspondingto position 519 (H) in SEQ ID NO: 1 is P. In one embodiment, such Sprotein variant comprises SARS-CoV-2 S protein or a naturally occurringvariant thereof wherein the amino acid corresponding to position 614 (D)in SEQ ID NO: 1 is G.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant such as SARS-CoV-2 S protein variant, in particular naturallyoccurring S protein variant comprising a mutation at a positioncorresponding to position 501 (N) in SEQ ID NO: 1. In one embodiment,the amino acid corresponding to position 501 (N) in SEQ ID NO: 1 is Y.

Said S protein variant comprising a mutation at a position correspondingto position 501 (N) in SEQ ID NO: 1 may comprise one or more furthermutations. Such one or more further mutations may be selected frommutations at positions corresponding to the following positions in SEQID NO: 1:69 (H), 70 (V), 144 (Y), 570 (A), 614 (D), 681 (P), 716 (T),982 (S), 1118 (D), 80 (D), 215 (D), 484 (E), 701 (A), 18 (L), 246 (R),417 (K), 242 (L), 243 (A), and 244 (L). In one embodiment, the aminoacid corresponding to position 69 (H) in SEQ ID NO: 1 is deleted. In oneembodiment, the amino acid corresponding to position 70 (V) in SEQ IDNO: 1 is deleted. In one embodiment, the amino acid corresponding toposition 144 (Y) in SEQ ID NO: 1 is deleted. In one embodiment, theamino acid corresponding to position 570 (A) in SEQ ID NO: 1 is D. Inone embodiment, the amino acid corresponding to position 614 (D) in SEQID NO: 1 is G. In one embodiment, the amino acid corresponding toposition 681 (P) in SEQ ID NO: 1 is H. In one embodiment, the amino acidcorresponding to position 716 (T) in SEQ ID NO: 1 is I. In oneembodiment, the amino acid corresponding to position 982 (S) in SEQ IDNO: 1 is A. In one embodiment, the amino acid corresponding to position1118 (D) in SEQ ID NO: 1 is H. In one embodiment, the amino acidcorresponding to position 80 (D) in SEQ ID NO: 1 is A. In oneembodiment, the amino acid corresponding to position 215 (D) in SEQ IDNO: 1 is G. In one embodiment, the amino acid corresponding to position484 (E) in SEQ ID NO: 1 is K. In one embodiment, the amino acidcorresponding to position 701 (A) in SEQ ID NO: 1 is V. In oneembodiment, the amino acid corresponding to position 18 (L) in SEQ IDNO: 1 is F. In one embodiment, the amino acid corresponding to position246 (R) in SEQ ID NO: 1 is I. In one embodiment, the amino acidcorresponding to position 417 (K) in SEQ ID NO: 1 is N. In oneembodiment, the amino acid corresponding to position 242 (L) in SEQ IDNO: 1 is deleted. In one embodiment, the amino acid corresponding toposition 243 (A) in SEQ ID NO: 1 is deleted. In one embodiment, theamino acid corresponding to position 244 (L) in SEQ ID NO: 1 is deleted.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targetsVOC-202012/01.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant comprising the following mutations at positions corresponding tothe following positions in SEQ ID NO: 1: deletion 69-70, deletion 144,N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets 501.V2.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant comprising the following mutations at positions corresponding tothe following positions in SEQ ID NO: 1: D80A, D215G, E484K, N501Y andA701V, and optionally: L18F, R246I, K417N, and deletion 242-244. Said Sprotein variant may also comprise a D->G mutation at a positioncorresponding to position 614 in SEQ ID NO: 1.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant such as SARS-CoV-2 S protein variant, in particular naturallyoccurring S protein variant comprising a deletion at a positioncorresponding to positions 69 (H) and 70 (V) in SEQ ID NO: 1.

In some embodiments, a S protein variant comprising a deletion at aposition corresponding to positions 69 (H) and 70 (V) in SEQ ID NO: 1may comprise one or more further mutations. Such one or more furthermutations may be selected from mutations at positions corresponding tothe following positions in SEQ ID NO: 1:144 (Y), 501 (N), 570 (A), 614(D), 681 (P), 716 (T), 982 (S), 1118 (D), 80 (D), 215 (D), 484 (E), 701(A), 18 (L), 246 (R), 417 (K), 242 (L), 243 (A), 244 (L), 453 (Y), 692(I), 1147 (S), and 1229 (M). In one embodiment, the amino acidcorresponding to position 144 (Y) in SEQ ID NO: 1 is deleted. In oneembodiment, the amino acid corresponding to position 501 (N) in SEQ IDNO: 1 is Y. In one embodiment, the amino acid corresponding to position570 (A) in SEQ ID NO: 1 is D. In one embodiment, the amino acidcorresponding to position 614 (D) in SEQ ID NO: 1 is G. In oneembodiment, the amino acid corresponding to position 681 (P) in SEQ IDNO: 1 is H. In one embodiment, the amino acid corresponding to position716 (T) in SEQ ID NO: 1 is I. In one embodiment, the amino acidcorresponding to position 982 (S) in SEQ ID NO: 1 is A. In oneembodiment, the amino acid corresponding to position 1118 (D) in SEQ IDNO: 1 is H. In one embodiment, the amino acid corresponding to position80 (D) in SEQ ID NO: 1 is A. In one embodiment, the amino acidcorresponding to position 215 (D) in SEQ ID NO: 1 is G. In oneembodiment, the amino acid corresponding to position 484 (E) in SEQ IDNO: 1 is K. In one embodiment, the amino acid corresponding to position701 (A) in SEQ ID NO: 1 is V. In one embodiment, the amino acidcorresponding to position 18 (L) in SEQ ID NO: 1 is F. In oneembodiment, the amino acid corresponding to position 246 (R) in SEQ IDNO: 1 is I. In one embodiment, the amino acid corresponding to position417 (K) in SEQ ID NO: 1 is N. In one embodiment, the amino acidcorresponding to position 242 (L) in SEQ ID NO: 1 is deleted. In oneembodiment, the amino acid corresponding to position 243 (A) in SEQ IDNO: 1 is deleted. In one embodiment, the amino acid corresponding toposition 244 (L) in SEQ ID NO: 1 is deleted. In one embodiment, theamino acid corresponding to position 453 (Y) in SEQ ID NO: 1 is F. Inone embodiment, the amino acid corresponding to position 692 (I) in SEQID NO: 1 is V. In one embodiment, the amino acid corresponding toposition 1147 (S) in SEQ ID NO: 1 is L. In one embodiment, the aminoacid corresponding to position 1229 (M) in SEQ ID NO: 1 is I.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targetsVOC-202012/01.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant comprising the following mutations at positions corresponding tothe following positions in SEQ ID NO: 1: deletion 69-70, deletion 144,N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets “Cluster 5”.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant comprising the following mutations at positions corresponding tothe following positions in SEQ ID NO: 1: deletion 69-70, Y453F, 1692V,M1229I, and optionally S1147L.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant such as SARS-CoV-2 S protein variant, in particular naturallyoccurring S protein variant comprising a mutation at a positioncorresponding to position 614 (D) in SEQ ID NO: 1. In one embodiment,the amino acid corresponding to position 614 (D) in SEQ ID NO: 1 is G.

In some embodiments, a S protein variant comprising a mutation at aposition corresponding to position 614 (D) in SEQ ID NO: 1 may compriseone or more further mutations. Such one or more further mutations may beselected from mutations at positions corresponding to the followingpositions in SEQ ID NO: 1:69 (H), 70 (V), 144 (Y), 501 (N), 570 (A), 681(P), 716 (T), 982 (S), 1118 (D), 80 (D), 215 (D), 484 (E), 701 (A), 18(L), 246 (R), 417 (K), 242 (L), 243 (A), 244 (L), 453 (Y), 692 (I), 1147(S), and 1229 (M). In one embodiment, the amino acid corresponding toposition 69 (H) in SEQ ID NO: 1 is deleted. In one embodiment, the aminoacid corresponding to position 70 (V) in SEQ ID NO: 1 is deleted. In oneembodiment, the amino acid corresponding to position 144 (Y) in SEQ IDNO: 1 is deleted. In one embodiment, the amino acid corresponding toposition 501 (N) in SEQ ID NO: 1 is Y. In one embodiment, the amino acidcorresponding to position 570 (A) in SEQ ID NO: 1 is D. In oneembodiment, the amino acid corresponding to position 681 (P) in SEQ IDNO: 1 is H. In one embodiment, the amino acid corresponding to position716 (T) in SEQ ID NO: 1 is I. In one embodiment, the amino acidcorresponding to position 982 (S) in SEQ ID NO: 1 is A. In oneembodiment, the amino acid corresponding to position 1118 (D) in SEQ IDNO: 1 is H. In one embodiment, the amino acid corresponding to position80 (D) in SEQ ID NO: 1 is A. In one embodiment, the amino acidcorresponding to position 215 (D) in SEQ ID NO: 1 is G. In oneembodiment, the amino acid corresponding to position 484 (E) in SEQ IDNO: 1 is K. In one embodiment, the amino acid corresponding to position701 (A) in SEQ ID NO: 1 is V. In one embodiment, the amino acidcorresponding to position 18 (L) in SEQ ID NO: 1 is F. In oneembodiment, the amino acid corresponding to position 246 (R) in SEQ IDNO: 1 is I. In one embodiment, the amino acid corresponding to position417 (K) in SEQ ID NO: 1 is N. In one embodiment, the amino acidcorresponding to position 242 (L) in SEQ ID NO: 1 is deleted. In oneembodiment, the amino acid corresponding to position 243 (A) in SEQ IDNO: 1 is deleted. In one embodiment, the amino acid corresponding toposition 244 (L) in SEQ ID NO: 1 is deleted. In one embodiment, theamino acid corresponding to position 453 (Y) in SEQ ID NO: 1 is F. Inone embodiment, the amino acid corresponding to position 692 (I) in SEQID NO: 1 is V. In one embodiment, the amino acid corresponding toposition 1147 (S) in SEQ ID NO: 1 is L. In one embodiment, the aminoacid corresponding to position 1229 (M) in SEQ ID NO: 1 is I.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targetsVOC-202012/01.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant comprising the following mutations at positions corresponding tothe following positions in SEQ ID NO: 1: deletion 69-70, deletion 144,N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant comprising the following mutations at positions corresponding tothe following positions in SEQ ID NO: 1: D80A, D215G, E484K, N501Y,D614G and A701V, and optionally: L18F, R246I, K417N, and deletion242-244.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant such as SARS-CoV-2 S protein variant, in particular naturallyoccurring S protein variant comprising a mutation at positionscorresponding to positions 501 (N) and 614 (D) in SEQ ID NO: 1. In oneembodiment, the amino acid corresponding to position 501 (N) in SEQ IDNO: 1 is Y and the amino acid corresponding to position 614 (D) in SEQID NO: 1 is G.

In some embodiments, a S protein variant comprising a mutation atpositions corresponding to positions 501 (N) and 614 (D) in SEQ ID NO: 1may comprise one or more further mutations. Such one or more furthermutations may be selected from mutations at positions corresponding tothe following positions in SEQ ID NO: 1:69 (H), 70 (V), 144 (Y), 570(A), 681 (P), 716 (T), 982 (S), 1118 (D), 80 (D), 215 (D), 484 (E), 701(A), 18 (L), 246 (R), 417 (K), 242 (L), 243 (A), 244 (L), 453 (Y), 692(I), 1147 (S), and 1229 (M). In one embodiment, the amino acidcorresponding to position 69 (H) in SEQ ID NO: 1 is deleted. In oneembodiment, the amino acid corresponding to position 70 (V) in SEQ IDNO: 1 is deleted. In one embodiment, the amino acid corresponding toposition 144 (Y) in SEQ ID NO: 1 is deleted. In one embodiment, theamino acid corresponding to position 570 (A) in SEQ ID NO: 1 is D. Inone embodiment, the amino acid corresponding to position 681 (P) in SEQID NO: 1 is H. In one embodiment, the amino acid corresponding toposition 716 (T) in SEQ ID NO: 1 is I. In one embodiment, the amino acidcorresponding to position 982 (S) in SEQ ID NO: 1 is A. In oneembodiment, the amino acid corresponding to position 1118 (D) in SEQ IDNO: 1 is H. In one embodiment, the amino acid corresponding to position80 (D) in SEQ ID NO: 1 is A. In one embodiment, the amino acidcorresponding to position 215 (D) in SEQ ID NO: 1 is G. In oneembodiment, the amino acid corresponding to position 484 (E) in SEQ IDNO: 1 is K. In one embodiment, the amino acid corresponding to position701 (A) in SEQ ID NO: 1 is V. In one embodiment, the amino acidcorresponding to position 18 (L) in SEQ ID NO: 1 is F. In oneembodiment, the amino acid corresponding to position 246 (R) in SEQ IDNO: 1 is I. In one embodiment, the amino acid corresponding to position417 (K) in SEQ ID NO: 1 is N. In one embodiment, the amino acidcorresponding to position 242 (L) in SEQ ID NO: 1 is deleted. In oneembodiment, the amino acid corresponding to position 243 (A) in SEQ IDNO: 1 is deleted. In one embodiment, the amino acid corresponding toposition 244 (L) in SEQ ID NO: 1 is deleted. In one embodiment, theamino acid corresponding to position 453 (Y) in SEQ ID NO: 1 is F. Inone embodiment, the amino acid corresponding to position 692 (I) in SEQID NO: 1 is V. In one embodiment, the amino acid corresponding toposition 1147 (S) in SEQ ID NO: 1 is L. In one embodiment, the aminoacid corresponding to position 1229 (M) in SEQ ID NO: 1 is I.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targetsVOC-202012/01.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant comprising the following mutations at positions corresponding tothe following positions in SEQ ID NO: 1: deletion 69-70, deletion 144,N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant comprising the following mutations at positions corresponding tothe following positions in SEQ ID NO: 1: D80A, D215G, E484K, N501Y,D614G and A701V, and optionally: L18F, R246I, K417N, and deletion242-244.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant such as SARS-CoV-2 S protein variant, in particular naturallyoccurring S protein variant comprising a mutation at a positioncorresponding to position 484 (E) in SEQ ID NO: 1. In one embodiment,the amino acid corresponding to position 484 (E) in SEQ ID NO: 1 is K.

In some embodiments, a S protein variant comprising a mutation at aposition corresponding to position 484 (E) in SEQ ID NO: 1 may compriseone or more further mutations. Such one or more further mutations may beselected from mutations at positions corresponding to the followingpositions in SEQ ID NO: 1:69 (H), 70 (V), 144 (Y), 501 (N), 570 (A), 614(D), 681 (P), 716 (T), 982 (S), 1118 (D), 80 (D), 215 (D), 701 (A), 18(L), 246 (R), 417 (K), 242 (L), 243 (A), 244 (L), 453 (Y), 692 (I), 1147(S), 1229 (M), 20 (T), 26 (P), 138 (D), 190 (R), 417 (K), 655 (H), 1027(T), and 1176 (V). In one embodiment, the amino acid corresponding toposition 69 (H) in SEQ ID NO: 1 is deleted. In one embodiment, the aminoacid corresponding to position 70 (V) in SEQ ID NO: 1 is deleted. In oneembodiment, the amino acid corresponding to position 144 (Y) in SEQ IDNO: 1 is deleted. In one embodiment, the amino acid corresponding toposition 501 (N) in SEQ ID NO: 1 is Y. In one embodiment, the amino acidcorresponding to position 570 (A) in SEQ ID NO: 1 is D. In oneembodiment, the amino acid corresponding to position 614 (D) in SEQ IDNO: 1 is G. In one embodiment, the amino acid corresponding to position681 (P) in SEQ ID NO: 1 is H. In one embodiment, the amino acidcorresponding to position 716 (T) in SEQ ID NO: 1 is I. In oneembodiment, the amino acid corresponding to position 982 (S) in SEQ IDNO: 1 is A. In one embodiment, the amino acid corresponding to position1118 (D) in SEQ ID NO: 1 is H. In one embodiment, the amino acidcorresponding to position 80 (D) in SEQ ID NO: 1 is A. In oneembodiment, the amino acid corresponding to position 215 (D) in SEQ IDNO: 1 is G. In one embodiment, the amino acid corresponding to position701 (A) in SEQ ID NO: 1 is V. In one embodiment, the amino acidcorresponding to position 18 (L) in SEQ ID NO: 1 is F. In oneembodiment, the amino acid corresponding to position 246 (R) in SEQ IDNO: 1 is I. In one embodiment, the amino acid corresponding to position417 (K) in SEQ ID NO: 1 is N. In one embodiment, the amino acidcorresponding to position 242 (L) in SEQ ID NO: 1 is deleted. In oneembodiment, the amino acid corresponding to position 243 (A) in SEQ IDNO: 1 is deleted. In one embodiment, the amino acid corresponding toposition 244 (L) in SEQ ID NO: 1 is deleted. In one embodiment, theamino acid corresponding to position 453 (Y) in SEQ ID NO: 1 is F. Inone embodiment, the amino acid corresponding to position 692 (I) in SEQID NO: 1 is V. In one embodiment, the amino acid corresponding toposition 1147 (S) in SEQ ID NO: 1 is L. In one embodiment, the aminoacid corresponding to position 1229 (M) in SEQ ID NO: 1 is I. In oneembodiment, the amino acid corresponding to position 20 (T) in SEQ IDNO: 1 is N. In one embodiment, the amino acid corresponding to position26 (P) in SEQ ID NO: 1 is S. In one embodiment, the amino acidcorresponding to position 138 (D) in SEQ ID NO: 1 is Y. In oneembodiment, the amino acid corresponding to position 190 (R) in SEQ IDNO: 1 is S. In one embodiment, the amino acid corresponding to position417 (K) in SEQ ID NO: 1 is T. In one embodiment, the amino acidcorresponding to position 655 (H) in SEQ ID NO: 1 is Y. In oneembodiment, the amino acid corresponding to position 1027 (T) in SEQ IDNO: 1 is I. In one embodiment, the amino acid corresponding to position1176 (V) in SEQ ID NO: 1 is F.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets 501.V2.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant comprising the following mutations at positions corresponding tothe following positions in SEQ ID NO: 1: D80A, D215G, E484K, N501Y andA701V, and optionally: L18F, R246I, K417N, and deletion 242-244. Said Sprotein variant may also comprise a D->G mutation at a positioncorresponding to position 614 in SEQ ID NO: 1.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets “B.1.1.28”.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets “B.1.1.248”.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant comprising the following mutations at positions corresponding tothe following positions in SEQ ID NO: 1: L18F, T20N, P26S, D138Y, R190S,K417T, E484K, N501Y, H655Y, T1027I, and V1176F.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant such as SARS-CoV-2 S protein variant, in particular naturallyoccurring S protein variant comprising a mutation at positionscorresponding to positions 501 (N) and 484 (E) in SEQ ID NO: 1. In oneembodiment, the amino acid corresponding to position 501 (N) in SEQ IDNO: 1 is Y and the amino acid corresponding to position 484 (E) in SEQID NO: 1 is K.

In some embodiments, a S protein variant comprising a mutation atpositions corresponding to positions 501 (N) and 484 (E) in SEQ ID NO: 1may comprise one or more further mutations.

Such one or more further mutations may be selected from mutations atpositions corresponding to the following positions in SEQ ID NO: 1:69(H), 70 (V), 144 (Y), 570 (A), 614 (D), 681 (P), 716 (T), 982 (S), 1118(D), 80 (D), 215 (D), 701 (A), 18 (L), 246 (R), 417 (K), 242 (L), 243(A), 244 (L), 453 (Y), 692 (I), 1147 (S), 1229 (M), 20 (T), 26 (P), 138(D), 190 (R), 417 (K), 655 (H), 1027 (T), and 1176 (V). In oneembodiment, the amino acid corresponding to position 69 (H) in SEQ IDNO: 1 is deleted. In one embodiment, the amino acid corresponding toposition 70 (V) in SEQ ID NO: 1 is deleted. In one embodiment, the aminoacid corresponding to position 144 (Y) in SEQ ID NO: 1 is deleted. Inone embodiment, the amino acid corresponding to position 570 (A) in SEQID NO: 1 is D. In one embodiment, the amino acid corresponding toposition 614 (D) in SEQ ID NO: 1 is G. In one embodiment, the amino acidcorresponding to position 681 (P) in SEQ ID NO: 1 is H. In oneembodiment, the amino acid corresponding to position 716 (T) in SEQ IDNO: 1 is I. In one embodiment, the amino acid corresponding to position982 (S) in SEQ ID NO: 1 is A. In one embodiment, the amino acidcorresponding to position 1118 (D) in SEQ ID NO: 1 is H. In oneembodiment, the amino acid corresponding to position 80 (D) in SEQ IDNO: 1 is A. In one embodiment, the amino acid corresponding to position215 (D) in SEQ ID NO: 1 is G. In one embodiment, the amino acidcorresponding to position 701 (A) in SEQ ID NO: 1 is V. In oneembodiment, the amino acid corresponding to position 18 (L) in SEQ IDNO: 1 is F. In one embodiment, the amino acid corresponding to position246 (R) in SEQ ID NO: 1 is I. In one embodiment, the amino acidcorresponding to position 417 (K) in SEQ ID NO: 1 is N. In oneembodiment, the amino acid corresponding to position 242 (L) in SEQ IDNO: 1 is deleted. In one embodiment, the amino acid corresponding toposition 243 (A) in SEQ ID NO: 1 is deleted. In one embodiment, theamino acid corresponding to position 244 (L) in SEQ ID NO: 1 is deleted.In one embodiment, the amino acid corresponding to position 453 (Y) inSEQ ID NO: 1 is F. In one embodiment, the amino acid corresponding toposition 692 (I) in SEQ ID NO: 1 is V. In one embodiment, the amino acidcorresponding to position 1147 (S) in SEQ ID NO: 1 is L. In oneembodiment, the amino acid corresponding to position 1229 (M) in SEQ IDNO: 1 is I. In one embodiment, the amino acid corresponding to position20 (T) in SEQ ID NO: 1 is N. In one embodiment, the amino acidcorresponding to position 26 (P) in SEQ ID NO: 1 is S. In oneembodiment, the amino acid corresponding to position 138 (D) in SEQ IDNO: 1 is Y. In one embodiment, the amino acid corresponding to position190 (R) in SEQ ID NO: 1 is S. In one embodiment, the amino acidcorresponding to position 417 (K) in SEQ ID NO: 1 is T. In oneembodiment, the amino acid corresponding to position 655 (H) in SEQ IDNO: 1 is Y. In one embodiment, the amino acid corresponding to position1027 (T) in SEQ ID NO: 1 is I. In one embodiment, the amino acidcorresponding to position 1176 (V) in SEQ ID NO: 1 is F.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets 501.V2.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant comprising the following mutations at positions corresponding tothe following positions in SEQ ID NO: 1: D80A, D215G, E484K, N501Y andA701V, and optionally: L18F, R246I, K417N, and deletion 242-244. Said Sprotein variant may also comprise a D->G mutation at a positioncorresponding to position 614 in SEQ ID NO: 1.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets “B.1.1.248”.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant comprising the following mutations at positions corresponding tothe following positions in SEQ ID NO: 1: L18F, T20N, P26S, D138Y, R190S,K417T, E484K, N501Y, H655Y, T1027I, and V1176F.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant such as SARS-CoV-2 S protein variant, in particular naturallyoccurring S protein variant comprising a mutation at positionscorresponding to positions 501 (N), 484 (E) and 614 (D) in SEQ ID NO: 1.In one embodiment, the amino acid corresponding to position 501 (N) inSEQ ID NO: 1 is Y, the amino acid corresponding to position 484 (E) inSEQ ID NO: 1 is K and the amino acid corresponding to position 614 (D)in SEQ ID NO: 1 is G.

In some embodiments, a S protein variant comprising a mutation atpositions corresponding to positions 501 (N), 484 (E) and 614 (D) in SEQID NO: 1 may comprise one or more further mutations. Such one or morefurther mutations may be selected from mutations at positionscorresponding to the following positions in SEQ ID NO: 1:69 (H), 70 (V),144 (Y), 570 (A), 681 (P), 716 (T), 982 (S), 1118 (D), 80 (D), 215 (D),701 (A), 18 (L), 246 (R), 417 (K), 242 (L), 243 (A), 244 (L), 453 (Y),692 (I), 1147 (S), 1229 (M), 20 (T), 26 (P), 138 (D), 190 (R), 417 (K),655 (H), 1027 (T), and 1176 (V). In one embodiment, the amino acidcorresponding to position 69 (H) in SEQ ID NO: 1 is deleted. In oneembodiment, the amino acid corresponding to position 70 (V) in SEQ IDNO: 1 is deleted. In one embodiment, the amino acid corresponding toposition 144 (Y) in SEQ ID NO: 1 is deleted. In one embodiment, theamino acid corresponding to position 570 (A) in SEQ ID NO: 1 is D. Inone embodiment, the amino acid corresponding to position 681 (P) in SEQID NO: 1 is H. In one embodiment, the amino acid corresponding toposition 716 (T) in SEQ ID NO: 1 is I. In one embodiment, the amino acidcorresponding to position 982 (S) in SEQ ID NO: 1 is A. In oneembodiment, the amino acid corresponding to position 1118 (D) in SEQ IDNO: 1 is H. In one embodiment, the amino acid corresponding to position80 (D) in SEQ ID NO: 1 is A. In one embodiment, the amino acidcorresponding to position 215 (D) in SEQ ID NO: 1 is G. In oneembodiment, the amino acid corresponding to position 701 (A) in SEQ IDNO: 1 is V. In one embodiment, the amino acid corresponding to position18 (L) in SEQ ID NO: 1 is F. In one embodiment, the amino acidcorresponding to position 246 (R) in SEQ ID NO: 1 is I. In oneembodiment, the amino acid corresponding to position 417 (K) in SEQ IDNO: 1 is N. In one embodiment, the amino acid corresponding to position242 (L) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acidcorresponding to position 243 (A) in SEQ ID NO: 1 is deleted. In oneembodiment, the amino acid corresponding to position 244 (L) in SEQ IDNO: 1 is deleted. In one embodiment, the amino acid corresponding toposition 453 (Y) in SEQ ID NO: 1 is F. In one embodiment, the amino acidcorresponding to position 692 (I) in SEQ ID NO: 1 is V. In oneembodiment, the amino acid corresponding to position 1147 (S) in SEQ IDNO: 1 is L. In one embodiment, the amino acid corresponding to position1229 (M) in SEQ ID NO: 1 is I. In one embodiment, the amino acidcorresponding to position 20 (T) in SEQ ID NO: 1 is N. In oneembodiment, the amino acid corresponding to position 26 (P) in SEQ IDNO: 1 is S. In one embodiment, the amino acid corresponding to position138 (D) in SEQ ID NO: 1 is Y. In one embodiment, the amino acidcorresponding to position 190 (R) in SEQ ID NO: 1 is S. In oneembodiment, the amino acid corresponding to position 417 (K) in SEQ IDNO: 1 is T. In one embodiment, the amino acid corresponding to position655 (H) in SEQ ID NO: 1 is Y. In one embodiment, the amino acidcorresponding to position 1027 (T) in SEQ ID NO: 1 is I. In oneembodiment, the amino acid corresponding to position 1176 (V) in SEQ IDNO: 1 is F.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant comprising the following mutations at positions corresponding tothe following positions in SEQ ID NO: 1: D80A, D215G, E484K, N501Y,A701V, and D614G, and optionally: L18F, R246I, K417N, and deletion242-244.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant such as SARS-CoV-2 S protein variant, in particular naturallyoccurring S protein variant comprising a deletion at a positioncorresponding to positions 242 (L), 243 (A) and 244 (L) in SEQ ID NO: 1.

In some embodiments, a S protein variant comprising a deletion at aposition corresponding to positions 242 (L), 243 (A) and 244 (L) in SEQID NO: 1 may comprise one or more further mutations. Such one or morefurther mutations may be selected from mutations at positionscorresponding to the following positions in SEQ ID NO: 1:69 (H), 70 (V),144 (Y), 501 (N), 570 (A), 614 (D), 681 (P), 716 (T), 982 (S), 1118 (D),80 (D), 215 (D), 484 (E), 701 (A), 18 (L), 246 (R), 417 (K), 453 (Y),692 (I), 1147 (S), 1229 (M), 20 (T), 26 (P), 138 (D), 190 (R), 417 (K),655 (H), 1027 (T), and 1176 (V). In one embodiment, the amino acidcorresponding to position 69 (H) in SEQ ID NO: 1 is deleted. In oneembodiment, the amino acid corresponding to position 70 (V) in SEQ IDNO: 1 is deleted. In one embodiment, the amino acid corresponding toposition 144 (Y) in SEQ ID NO: 1 is deleted. In one embodiment, theamino acid corresponding to position 501 (N) in SEQ ID NO: 1 is Y. Inone embodiment, the amino acid corresponding to position 570 (A) in SEQID NO: 1 is D. In one embodiment, the amino acid corresponding toposition 614 (D) in SEQ ID NO: 1 is G. In one embodiment, the amino acidcorresponding to position 681 (P) in SEQ ID NO: 1 is H. In oneembodiment, the amino acid corresponding to position 716 (T) in SEQ IDNO: 1 is I. In one embodiment, the amino acid corresponding to position982 (S) in SEQ ID NO: 1 is A. In one embodiment, the amino acidcorresponding to position 1118 (D) in SEQ ID NO: 1 is H. In oneembodiment, the amino acid corresponding to position 80 (D) in SEQ IDNO: 1 is A. In one embodiment, the amino acid corresponding to position215 (D) in SEQ ID NO: 1 is G. In one embodiment, the amino acidcorresponding to position 484 (E) in SEQ ID NO: 1 is K. In oneembodiment, the amino acid corresponding to position 701 (A) in SEQ IDNO: 1 is V. In one embodiment, the amino acid corresponding to position18 (L) in SEQ ID NO: 1 is F. In one embodiment, the amino acidcorresponding to position 246 (R) in SEQ ID NO: 1 is I. In oneembodiment, the amino acid corresponding to position 417 (K) in SEQ IDNO: 1 is N. In one embodiment, the amino acid corresponding to position453 (Y) in SEQ ID NO: 1 is F. In one embodiment, the amino acidcorresponding to position 692 (I) in SEQ ID NO: 1 is V. In oneembodiment, the amino acid corresponding to position 1147 (S) in SEQ IDNO: 1 is L. In one embodiment, the amino acid corresponding to position1229 (M) in SEQ ID NO: 1 is I. In one embodiment, the amino acidcorresponding to position 20 (T) in SEQ ID NO: 1 is N. In oneembodiment, the amino acid corresponding to position 26 (P) in SEQ IDNO: 1 is S. In one embodiment, the amino acid corresponding to position138 (D) in SEQ ID NO: 1 is Y. In one embodiment, the amino acidcorresponding to position 190 (R) in SEQ ID NO: 1 is S. In oneembodiment, the amino acid corresponding to position 417 (K) in SEQ IDNO: 1 is T. In one embodiment, the amino acid corresponding to position655 (H) in SEQ ID NO: 1 is Y. In one embodiment, the amino acidcorresponding to position 1027 (T) in SEQ ID NO: 1 is I. In oneembodiment, the amino acid corresponding to position 1176 (V) in SEQ IDNO: 1 is F.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets 501.V2.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant comprising the following mutations at positions corresponding tothe following positions in SEQ ID NO: 1: D80A, D215G, E484K, N501Y,A701V and deletion 242-244, and optionally: L18F, R246I, and K417N. SaidS protein variant may also comprise a D->G mutation at a positioncorresponding to position 614 in SEQ ID NO: 1.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant such as SARS-CoV-2 S protein variant, in particular naturallyoccurring S protein variant comprising a mutation at a positioncorresponding to position 417 (K) in SEQ ID NO: 1. In one embodiment,the amino acid corresponding to position 417 (K) in SEQ ID NO: 1 is N.In one embodiment, the amino acid corresponding to position 417 (K) inSEQ ID NO: 1 is T.

In some embodiments, a S protein variant comprising a mutation at aposition corresponding to position 417 (K) in SEQ ID NO: 1 may compriseone or more further mutations. Such one or more further mutations may beselected from mutations at positions corresponding to the followingpositions in SEQ ID NO: 1:69 (H), 70 (V), 144 (Y), 501 (N), 570 (A), 614(D), 681 (P), 716 (T), 982 (S), 1118 (D), 80 (D), 215 (D), 484 (E), 701(A), 18 (L), 246 (R), 242 (L), 243 (A), 244 (L), 453 (Y), 692 (I), 1147(S), 1229 (M), 20 (T), 26 (P), 138 (D), 190 (R), 655 (H), 1027 (T), and1176 (V). In one embodiment, the amino acid corresponding to position 69(H) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acidcorresponding to position 70 (V) in SEQ ID NO: 1 is deleted. In oneembodiment, the amino acid corresponding to position 144 (Y) in SEQ IDNO: 1 is deleted. In one embodiment, the amino acid corresponding toposition 501 (N) in SEQ ID NO: 1 is Y. In one embodiment, the amino acidcorresponding to position 570 (A) in SEQ ID NO: 1 is D. In oneembodiment, the amino acid corresponding to position 614 (D) in SEQ IDNO: 1 is G. In one embodiment, the amino acid corresponding to position681 (P) in SEQ ID NO: 1 is H. In one embodiment, the amino acidcorresponding to position 716 (T) in SEQ ID NO: 1 is I. In oneembodiment, the amino acid corresponding to position 982 (S) in SEQ IDNO: 1 is A. In one embodiment, the amino acid corresponding to position1118 (D) in SEQ ID NO: 1 is H. In one embodiment, the amino acidcorresponding to position 80 (D) in SEQ ID NO: 1 is A. In oneembodiment, the amino acid corresponding to position 215 (D) in SEQ IDNO: 1 is G. In one embodiment, the amino acid corresponding to position484 (E) in SEQ ID NO: 1 is K. In one embodiment, the amino acidcorresponding to position 701 (A) in SEQ ID NO: 1 is V. In oneembodiment, the amino acid corresponding to position 18 (L) in SEQ IDNO: 1 is F. In one embodiment, the amino acid corresponding to position246 (R) in SEQ ID NO: 1 is I. In one embodiment, the amino acidcorresponding to position 242 (L) in SEQ ID NO: 1 is deleted. In oneembodiment, the amino acid corresponding to position 243 (A) in SEQ IDNO: 1 is deleted. In one embodiment, the amino acid corresponding toposition 244 (L) in SEQ ID NO: 1 is deleted. In one embodiment, theamino acid corresponding to position 453 (Y) in SEQ ID NO: 1 is F. Inone embodiment, the amino acid corresponding to position 692 (I) in SEQID NO: 1 is V. In one embodiment, the amino acid corresponding toposition 1147 (S) in SEQ ID NO: 1 is L. In one embodiment, the aminoacid corresponding to position 1229 (M) in SEQ ID NO: 1 is I. In oneembodiment, the amino acid corresponding to position 20 (T) in SEQ IDNO: 1 is N. In one embodiment, the amino acid corresponding to position26 (P) in SEQ ID NO: 1 is S. In one embodiment, the amino acidcorresponding to position 138 (D) in SEQ ID NO: 1 is Y. In oneembodiment, the amino acid corresponding to position 190 (R) in SEQ IDNO: 1 is S. In one embodiment, the amino acid corresponding to position655 (H) in SEQ ID NO: 1 is Y. In one embodiment, the amino acidcorresponding to position 1027 (T) in SEQ ID NO: 1 is I. In oneembodiment, the amino acid corresponding to position 1176 (V) in SEQ IDNO: 1 is F.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets 501.V2.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant comprising the following mutations at positions corresponding tothe following positions in SEQ ID NO: 1: D80A, D215G, E484K, N501Y,A701V, and K417N, and optionally: L18F, R246I, and deletion 242-244.Said S protein variant may also comprise a D->G mutation at a positioncorresponding to position 614 in SEQ ID NO: 1.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets “B.1.1.248”.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant comprising the following mutations at positions corresponding tothe following positions in SEQ ID NO: 1: L18F, T20N, P26S, D138Y, R190S,K417T, E484K, N501Y, H655Y, T1027I, and V1176F.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant such as SARS-CoV-2 S protein variant, in particular naturallyoccurring S protein variant comprising a mutation at positionscorresponding to positions 417 (K) and 484 (E) and/or 501 (N) in SEQ IDNO: 1. In one embodiment, the amino acid corresponding to position 417(K) in SEQ ID NO: 1 is N, and the amino acid corresponding to position484 (E) in SEQ ID NO: 1 is K and/or the amino acid corresponding toposition 501 (N) in SEQ ID NO: 1 is Y. In one embodiment, the amino acidcorresponding to position 417 (K) in SEQ ID NO: 1 is T, and the aminoacid corresponding to position 484 (E) in SEQ ID NO: 1 is K and/or theamino acid corresponding to position 501 (N) in SEQ ID NO: 1 is Y.

In some embodiments, a S protein variant comprising a mutation atpositions corresponding to positions 417 (K) and 484 (E) and/or 501 (N)in SEQ ID NO: 1 may comprise one or more further mutations. Such one ormore further mutations may be selected from mutations at positionscorresponding to the following positions in SEQ ID NO: 1:69 (H), 70 (V),144 (Y), 570 (A), 614 (D), 681 (P), 716 (T), 982 (S), 1118 (D), 80 (D),215 (D), 701 (A), 18 (L), 246 (R), 242 (L), 243 (A), 244 (L), 453 (Y),692 (I), 1147 (S), 1229 (M), 20 (T), 26 (P), 138 (D), 190 (R), 655 (H),1027 (T), and 1176 (V). In one embodiment, the amino acid correspondingto position 69 (H) in SEQ ID NO: 1 is deleted. In one embodiment, theamino acid corresponding to position 70 (V) in SEQ ID NO: 1 is deleted.In one embodiment, the amino acid corresponding to position 144 (Y) inSEQ ID NO: 1 is deleted. In one embodiment, the amino acid correspondingto position 570 (A) in SEQ ID NO: 1 is D. In one embodiment, the aminoacid corresponding to position 614 (D) in SEQ ID NO: 1 is G. In oneembodiment, the amino acid corresponding to position 681 (P) in SEQ IDNO: 1 is H. In one embodiment, the amino acid corresponding to position716 (T) in SEQ ID NO: 1 is I. In one embodiment, the amino acidcorresponding to position 982 (S) in SEQ ID NO: 1 is A. In oneembodiment, the amino acid corresponding to position 1118 (D) in SEQ IDNO: 1 is H. In one embodiment, the amino acid corresponding to position80 (D) in SEQ ID NO: 1 is A. In one embodiment, the amino acidcorresponding to position 215 (D) in SEQ ID NO: 1 is G. In oneembodiment, the amino acid corresponding to position 701 (A) in SEQ IDNO: 1 is V. In one embodiment, the amino acid corresponding to position18 (L) in SEQ ID NO: 1 is F. In one embodiment, the amino acidcorresponding to position 246 (R) in SEQ ID NO: 1 is I. In oneembodiment, the amino acid corresponding to position 242 (L) in SEQ IDNO: 1 is deleted. In one embodiment, the amino acid corresponding toposition 243 (A) in SEQ ID NO: 1 is deleted. In one embodiment, theamino acid corresponding to position 244 (L) in SEQ ID NO: 1 is deleted.In one embodiment, the amino acid corresponding to position 453 (Y) inSEQ ID NO: 1 is F. In one embodiment, the amino acid corresponding toposition 692 (I) in SEQ ID NO: 1 is V. In one embodiment, the amino acidcorresponding to position 1147 (S) in SEQ ID NO: 1 is L. In oneembodiment, the amino acid corresponding to position 1229 (M) in SEQ IDNO: 1 is I. In one embodiment, the amino acid corresponding to position20 (T) in SEQ ID NO: 1 is N. In one embodiment, the amino acidcorresponding to position 26 (P) in SEQ ID NO: 1 is S. In oneembodiment, the amino acid corresponding to position 138 (D) in SEQ IDNO: 1 is Y. In one embodiment, the amino acid corresponding to position190 (R) in SEQ ID NO: 1 is S. In one embodiment, the amino acidcorresponding to position 655 (H) in SEQ ID NO: 1 is Y. In oneembodiment, the amino acid corresponding to position 1027 (T) in SEQ IDNO: 1 is I. In one embodiment, the amino acid corresponding to position1176 (V) in SEQ ID NO: 1 is F.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets 501.V2.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant comprising the following mutations at positions corresponding tothe following positions in SEQ ID NO: 1: D80A, D215G, E484K, N501Y,A701V, and K417N and optionally: L18F, R246I, and deletion 242-244. SaidS protein variant may also comprise a D->G mutation at a positioncorresponding to position 614 in SEQ ID NO: 1.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets “B.1.1.248”.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant comprising the following mutations at positions corresponding tothe following positions in SEQ ID NO: 1: L18F, T20N, P26S, D138Y, R190S,K417T, E484K, N501Y, H655Y, T1027I, and V1176F.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets the Omicron(B.1.1.529) variant.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant comprising at least 10, at least 15, at least 20, at least 21,at least 22, at least 23, at least 24, at least 25, at least 26, atleast 27, at least 28, at least 29, at least 30, at least 31, at least32, at least 33, at least 34, at least 35, at least 36, or at least 37of the following mutations: T547K, H655Y, D614G, N679K, P681H, N969K,S373P, S371L, N440K, G339D, G446S, N856K, N764K, K417N, D796Y, Q954H,T95I, A67V, L981F, S477N, G496S, T478K, Q498R, Q493R, E484A, N501Y,S375F, Y505H, V143del, H69del, V70del, N211del, L212I, ins214EPE, G142D,Y144del, Y145del, L141del, Y144F, Y145D, G142del, as compared to SEQ IDNO: 1.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant comprising at least 10, at least 15, at least 20, at least 21,at least 22, at least 23, at least 24, or all of the followingmutations: T547K, H655Y, D614G, N679K, P681H, N969K, S373P, S371L,N440K, G339D, G446S, N856K, N764K, K417N, D796Y, Q954H, T95I, A67V,L981F, S477N, G496S, T478K, Q498R, Q493R, E484A, as compared to SEQ IDNO: 1. Said S protein variant may include at least 1, at least 2, atleast 3, at least 4, at least 5, or all of the following mutations:N501Y, S375F, Y505H, V143del, H69del, V70del, as compared to SEQ ID NO:1 and/or may include at least 1, at least 2, at least 3, at least 4, atleast 5, or all of the following mutations: N211del, L212I, ins214EPE,G142D, Y144del, Y145del, as compared to SEQ ID NO: 1. In someembodiments, said S protein variant may include at least 1, at least 2,at least 3, or all of the following mutations: L141del, Y144F, Y145D,G142del, as compared to SEQ ID NO: 1.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant comprising at least 10, at least 15, at least 20, at least 21,at least 22, at least 23, at least 24, at least 25, at least 26, atleast 27, at least 28, at least 29, at least 30, at least 31, at least32, or at least 33 of the following mutations: A67V, Δ69-70, T95I,G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F,K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y,Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H,N969K, and L981F, as compared to SEQ ID NO: 1.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant comprising the following mutations:

A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D,S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R,G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K,D796Y, N856K, Q954H, N969K, and L981F, as compared to SEQ ID NO: 1.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an antibody response, in particular aneutralizing antibody response, in the subject that targets a S proteinvariant comprising the following mutations:

A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D,S371L, S373P, S375F, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y,Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H,N969K, and L981F, as compared to SEQ ID NO: 1.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an immune response (cellular and/orantibody response, in particular neutralizing antibody response) in thesubject that targets the Omicron (B.1.1.529) variant. In someembodiments, the methods and agents described herein followingadministration to a subject induce an immune response (cellular and/orantibody response, in particular neutralizing antibody response) in thesubject that targets a S protein variant comprising at least 10, atleast 15, at least 20, at least 21, at least 22, at least 23, at least24, at least 25, at least 26, at least 27, at least 28, at least 29, atleast 30, at least 31, at least 32, at least 33, at least 34, at least35, at least 36, or at least 37 of the following mutations: T547K,H655Y, D614G, N679K, P681H, N969K, S373P, S371L, N440K, G339D, G446S,N856K, N764K, K417N, D796Y, Q954H, T95I, A67V, L981F, 5477N, G496S,T478K, Q498R, Q493R, E484A, N501Y, S375F, Y505H, V143del, H69del,V70del, N211del, L212I, ins214EPE, G142D, Y144del, Y145del, 1141del,Y144F, Y145D, G142del, as compared to SEQ ID NO: 1.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an immune response (cellular and/orantibody response, in particular neutralizing antibody response) in thesubject that targets a S protein variant comprising at least 10, atleast 15, at least 20, at least 21, at least 22, at least 23, at least24, or all of the following mutations: T547K, H655Y, D614G, N679K,P681H, N969K, S373P, S371L, N440K, G339D, G446S, N856K, N764K, K417N,D796Y, Q954H, T95I, A67V, L981F, S477N, G496S, T478K, Q498R, Q493R,E484A, as compared to SEQ ID NO: 1. Said S protein variant may includeat least 1, at least 2, at least 3, at least 4, at least 5, or all ofthe following mutations: N501Y, S375F, Y505H, V143del, H69del, V70del,as compared to SEQ ID NO: 1 and/or may include at least 1, at least 2,at least 3, at least 4, at least 5, or all of the following mutations:N211del, L212I, ins214EPE, G142D, Y144del, Y145del, as compared to SEQID NO: 1. In some embodiments, said S protein variant may include atleast 1, at least 2, at least 3, or all of the following mutations:L141del, Y144F, Y145D, G142del, as compared to SEQ ID NO: 1.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an immune response (cellular and/orantibody response, in particular neutralizing antibody response) in thesubject that targets a S protein variant comprising at least 10, atleast 15, at least 20, at least 21, at least 22, at least 23, at least24, at least 25, at least 26, at least 27, at least 28, at least 29, atleast 30, at least 31, at least 32, or at least 33 of the followingmutations:

A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D,S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R,G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K,D796Y, N856K, Q954H, N969K, and L981F, as compared to SEQ ID NO: 1.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an immune response (cellular and/orantibody response, in particular neutralizing antibody response) in thesubject that targets a S protein variant comprising the followingmutations:

A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D,S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R,G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K,D796Y, N856K, Q954H, N969K, and L981F, as compared to SEQ ID NO: 1.

In some embodiments, the methods and agents described herein followingadministration to a subject induce an immune response (cellular and/orantibody response, in particular neutralizing antibody response) in thesubject that targets a S protein variant comprising the followingmutations:

A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D,S371L, S373P, S375F, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y,Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H,N969K, and L981F, as compared to SEQ ID NO: 1.

The term “amino acid corresponding to position . . . ” as used hereinrefers to an amino acid position number corresponding to an amino acidposition number in SARS-CoV-2 S protein, in particular the amino acidsequence shown in SEQ ID NO: 1. The phrase “as compared to SEQ ID NO: 1”is equivalent to “at positions corresponding to the following positionsin SEQ ID NO: 1”. Corresponding amino acid positions in othercoronavirus S protein variants such as SARS-CoV-2 S protein variants maybe found by alignment with SARS-CoV-2 S protein, in particular the aminoacid sequence shown in SEQ ID NO: 1. It is considered well-known in theart how to align a sequence or segment in a sequence and therebydetermine the corresponding position in a sequence to an amino acidposition according to the present disclosure. Standard sequencealignment programs such as ALIGN, ClustalW or similar, typically atdefault settings may be used.

In some embodiments, the panel of different S protein variants to whichan antibody response is targeted comprises at least 5, at least 10, atleast 15, or even more S protein variants selected from the groupconsisting of the Q321S, V341I, A348T, N354D, S359N, V367F, K378S,R408I, Q409E, A435S, K458R, I472V, G476S, V483A, Y508H, H519P and D614Gvariants described above. In some embodiments, the panel of different Sprotein variants to which an antibody response is targeted comprises allS protein variants from the group consisting of the Q321S, V341I, A348T,N354D, S359N, V367F, K378S, R408I, Q409E, A435S, K458R, I472V, G476S,V483A, Y508H, H519P and D614G variants described above.

In some embodiments, the panel of different S protein variants to whichan antibody response is targeted comprises at least 5, at least 10, atleast 15, or even more S protein variants selected from the groupconsisting of the Q321L, V341I, A348T, N354D, S359N, V367F, K378R,R408I, Q409E, A435S, N439K, K458R, I472V, G476S, S477N, V483A, Y508H,H519P and D614G variants described above. In some embodiments, the panelof different S protein variants to which an antibody response istargeted comprises all S protein variants from the group consisting ofthe Q321L, V341I, A348T, N354D, S359N, V367F, K378R, R408I, Q409E,A435S, N439K, K458R, I472V, G476S, S477N, V483A, Y508H, H519P and D614Gvariants described above.

In some embodiments, a SARS-CoV-2 S protein, an immunogenic variantthereof, or an immunogenic fragment of the SARS-CoV-2 S protein or theimmunogenic variant thereof, e.g., as encoded by the RNA describedherein, comprises one or more of the mutations described herein for Sprotein variants such as SARS-CoV-2 S protein variants, in particularnaturally occurring S protein variants. In one embodiment, a SARS-CoV-2S protein, an immunogenic variant thereof, or an immunogenic fragment ofthe SARS-CoV-2 S protein or the immunogenic variant thereof, e.g., asencoded by the RNA described herein, comprises a mutation at a positioncorresponding to position 501 (N) in SEQ ID NO: 1. In one embodiment,the amino acid corresponding to position 501 (N) in SEQ ID NO: 1 is Y.In some embodiments, a SARS-CoV-2 S protein, an immunogenic variantthereof, or an immunogenic fragment of the SARS-CoV-2 S protein or theimmunogenic variant thereof, e.g., as encoded by the RNA describedherein, comprises one or more mutations, such as all mutations, of aSARS-CoV-2 S protein of a SARS-CoV-2 variant selected from the groupconsisting of VOC-202012/01, 501.V2, Cluster 5 and B.1.1.248. In someembodiments, a SARS-CoV-2 S protein, an immunogenic variant thereof, oran immunogenic fragment of the SARS-CoV-2 S protein or the immunogenicvariant thereof, e.g., as encoded by the RNA described herein, comprisesan amino acid sequence with alanine substitution at position 80, glycinesubstitution at position 215, lysine substitution at position 484,tyrosine substitution at position 501, valine substitution at position701, phenylalanine substitution at position 18, isoleucine substitutionat position 246, asparagine substitution at position 417, glycinesubstitution at position 614, deletions at positions 242 to 244, andproline substitutions at positions 986 and 987 of SEQ ID NO:1.

In some embodiments, a SARS-CoV-2 S protein, an immunogenic variantthereof, or an immunogenic fragment of the SARS-CoV-2 S protein or theimmunogenic variant thereof, e.g., as encoded by the RNA describedherein, comprises at least 10, at least 15, at least 20, at least 21, atleast 22, at least 23, at least 24, at least 25, at least 26, at least27, at least 28, at least 29, at least 30, at least 31, at least 32, atleast 33, at least 34, at least 35, at least 36, or at least 37 of thefollowing mutations: T547K, H655Y, D614G, N679K, P681H, N969K, S373P,S371L, N440K, G339D, G446S, N856K, N764K, K417N, D796Y, Q954H, T95I,A67V, L981F, S477N, G496S, T478K, Q498R, Q493R, E484A, N501Y, S375F,Y505H, V143del, H69del, V70del, N211del, L212I, ins214EPE, G142D,Y144del, Y145del, L141del, Y144F, Y145D, G142del, as compared to SEQ IDNO: 1. In some embodiments, a SARS-CoV-2 S protein, an immunogenicvariant thereof, or an immunogenic fragment of the SARS-CoV-2 S proteinor the immunogenic variant thereof, e.g., as encoded by the RNAdescribed herein, comprising said mutations comprises K986P and V987P,as compared to SEQ ID NO: 1.

In some embodiments, a SARS-CoV-2 S protein, an immunogenic variantthereof, or an immunogenic fragment of the SARS-CoV-2 S protein or theimmunogenic variant thereof, e.g., as encoded by the RNA describedherein, comprises at least 10, at least 15, at least 20, at least 21, atleast 22, at least 23, at least 24, or all of the following mutations:T547K, H655Y, D614G, N679K, P681H, N969K, S373P, S371L, N440K, G339D,G446S, N856K, N764K, K417N, D796Y, Q954H, T95I, A67V, L981F, S477N,G496S, T478K, Q498R, Q493R, E484A, as compared to SEQ ID NO: 1. SaidSARs-CoV-2 S protein, variant, or fragment may include at least 1, atleast 2, at least 3, at least 4, at least 5, or all of the followingmutations: N501Y, S375F, Y505H, V143del, H69del, V70del, as compared toSEQ ID NO: 1 and/or may include at least 1, at least 2, at least 3, atleast 4, at least 5, or all of the following mutations: N211del, L212I,ins214EPE, G142D, Y144del, Y145del, as compared to SEQ ID NO: 1. In someembodiments, said SARs-CoV-2 S protein, variant, or fragment may includeat least 1, at least 2, at least 3, or all of the following mutations:L141del, Y144F, Y145D, G142del, as compared to SEQ ID NO: 1. In someembodiments, a SARS-CoV-2 S protein, an immunogenic variant thereof, oran immunogenic fragment of the SARS-CoV-2 S protein or the immunogenicvariant thereof, e.g., as encoded by the RNA described herein,comprising said mutations comprises K986P and V987P, as compared to SEQID NO: 1.

In some embodiments, a SARS-CoV-2 S protein, an immunogenic variantthereof, or an immunogenic fragment of the SARS-CoV-2 S protein or theimmunogenic variant thereof, e.g., as encoded by the RNA describedherein, comprises at least 10, at least 15, at least 20, at least 21, atleast 22, at least 23, at least 24, at least 25, at least 26, at least27, at least 28, at least 29, at least 30, at least 31, at least 32, orat least 33 of the following mutations: A67V, Δ69-70, T95I, G142D,Δ143-145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N,N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H,T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K,and L981F, as compared to SEQ ID NO: 1. In some embodiments, aSARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenicfragment of the SARS-CoV-2 S protein or the immunogenic variant thereof,e.g., as encoded by the RNA described herein, comprising said mutationscomprises K986P and V987P, as compared to SEQ ID NO: 1.

In some embodiments, a SARS-CoV-2 S protein, an immunogenic variantthereof, or an immunogenic fragment of the SARS-CoV-2 S protein or theimmunogenic variant thereof, e.g., as encoded by the RNA describedherein, comprises the following mutations: A67V, Δ69-70, T95I, G142D,Δ143-145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N,N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H,T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K,and L981F, as compared to SEQ ID NO: 1.

In some embodiments, a SARS-CoV-2 S protein, an immunogenic variantthereof, or an immunogenic fragment of the SARS-CoV-2 S protein or theimmunogenic variant thereof, e.g., as encoded by the RNA describedherein, comprising said mutations comprises K986P and V987P, as comparedto SEQ ID NO: 1.

In some embodiments, a SARS-CoV-2 S protein, an immunogenic variantthereof, or an immunogenic fragment of the SARS-CoV-2 S protein or theimmunogenic variant thereof, e.g., as encoded by the RNA describedherein, comprises the following mutations: A67V, Δ69-70, T95I, G142D,Δ143-145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F, S477N,T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y,N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F, as comparedto SEQ ID NO: 1.

In some embodiments, a SARS-CoV-2 S protein, an immunogenic variantthereof, or an immunogenic fragment of the SARS-CoV-2 S protein or theimmunogenic variant thereof, e.g., as encoded by the RNA describedherein, comprising said mutations comprises K986P and V987P, as comparedto SEQ ID NO: 1.

In some embodiments, a SARS-CoV-2 S protein, an immunogenic variantthereof, or an immunogenic fragment of the SARS-CoV-2 S protein or theimmunogenic variant thereof, e.g., as encoded by the RNA describedherein, comprises the following mutations:

A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D,S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R,G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K,D796Y, N856K, Q954H, N969K, L981F, K986P and V987P, as compared to SEQID NO: 1.

In some embodiments, a SARS-CoV-2 S protein, an immunogenic variantthereof, or an immunogenic fragment of the SARS-CoV-2 S protein or theimmunogenic variant thereof, e.g., as encoded by the RNA describedherein, comprises the following mutations:

A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D,S371L, S373P, S375F, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y,Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H,N969K, L981F, K986P and V987P, as compared to SEQ ID NO: 1.

In some embodiments, a SARS-CoV-2 S protein, an immunogenic variantthereof, or an immunogenic fragment of the SARS-CoV-2 S protein or theimmunogenic variant thereof, e.g., as encoded by the RNA describedherein, comprises the following mutations:

In some embodiments, the spike changes in Omicron BA.2 variant includeT19I, A24-26, A27S, G142D, V213G, G339D, S371F, S373P, S375F, T376A,D405N, R408S, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y,Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, K986P,and V987P, as compared to SEQ ID NO: 1.

In some embodiments, a SARS-CoV-2 S protein, an immunogenic variantthereof, or an immunogenic fragment of the SARS-CoV-2 S protein or theimmunogenic variant thereof, e.g., as encoded by the RNA describedherein, comprises the following mutations:

T19I, A24-26, A27S, Δ69/70, G142D, V213G, G339D, S371F, S373P, S375F,T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V,Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H,N969K, K986P, and V987P, as compared to SEQ ID NO: 1. In someembodiments, administration of a variant specific vaccine (e.g., avariant specific vaccine disclosed herein) may result in an improvedimmune response in a patient as compared to administration of vaccineencoding or comprising a SARS-CoV-2 S protein from a Wuhan strain, or animmunogenic fragment thereof. In some embodiments, administration of avariant-specific vaccine may result in induction of a broader immuneresponse in a subject as compared to a patient administered a vaccinecomprising or encoding a SARS-CoV-2 S protein from a Wuhan strain (or animmunogenic fragment thereof) (e.g., induce a stronger neutralizationresponse against a greater number of SARS-CoV-2 variants and/or aneutralization response that recognizes epitopes in a greater number ofSARS-CoV-2 variants). In particular embodiments, a broader immuneresponse may be induced when a variant specific vaccine is administeredin combination with a vaccine comprising or encoding a SARS-CoV-2 Sprotein from a different variant or from a Wuhan strain (e.g., in someembodiments, a broader immune response may be induced when a variantspecific vaccine is administered in combination with a vaccinecomprising or encoding a SARS-CoV-2 S protein from a Wuhan strain or avaccine comprising or encoding a SARS-CoV-2 S protein comprisingmutations characteristic of a different SARS-CoV-2 variant). Forexample, a broader immune response may be induced when an RNA vaccineencoding a SARS-CoV-2 S protein from a Wuhan strain is administered incombination with an RNA vaccine encoding a SARS-CoV-2 S protein havingmutations characteristic of an Omicron variant. In another embodiment, abroader immune response may be induced when an RNA vaccine encoding aSARS-CoV-2 S protein comprising one or more mutations characteristic ofa delta variant is administered in combination with an RNA vaccineencoding a SARS-CoV-2 S protein comprising one or more mutationscharacteristic of an Omicron variant. In such embodiments, a “broader”immune response may be defined relative to a patient administered avaccine comprising or encoding a SARS-CoV-2 S protein from a singlevariant (e.g., an RNA vaccine encoding a SARS-CoV-2 S protein from aWuhan strain). Vaccines comprising or encoding S proteins from differentSARS-CoV-2 variants, or immunogenic fragments thereof, may beadministered in combination by administering at different time points(e.g., administering a vaccine encoding a SARS-CoV-2 S protein from aWuhan strain and a vaccine encoding a SARS-CoV-2 S protein having one ormore mutations characteristic of a variant strain at different timepoints, e.g., both administered as part of a primary regimen or part ofa booster regimen; or one is administered as part of a primary regimenwhile another is administered as part of a booster regimen). In someembodiments, vaccines comprising or encoding S proteins from differentSARS-CoV-2 variants, or immunogenic fragments thereof, may beadministered in combination by administering a multivalent vaccine(e.g., a composition comprising RNA encoding a SARS-CoV-2 S protein froma Wuhan strain and RNA encoding a SARS-CoV-2 S protein having mutationscharacteristic of an Omicron variant). In some embodiments, a variantspecific vaccine may induce a superior immune response (e.g., inducinghigher concentrations of neutralizing antibodies) against a variantagainst which the vaccine is specifically designed to immunize, and animmune response against one or more other variants. In some suchembodiments, an immune response against other variant(s) may becomparable to or higher than that as observed with a vaccine thatencodes or comprises a SARS-CoV-2 S protein from a Wuhan strain.

In some embodiments, the geometric mean ratio (GMR) or geometric meanfold rise (GMFR) of neutralization antibodies induced by a variantspecific vaccine is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,1.9, or 2.0 (e.g., 1.1 to 4, 1.1 to 3.5, 1.1 to 3, 1.5 to 3, or 1.1 to1.5) fold higher than that induced by a non-variant specific vaccine(e.g., as measured 1 day to 3 months after immunization, 7 days to 2months after administration, about 7 days, or about 1 month afteradministration).

In some embodiments, a SARS-CoV-2 S protein, an immunogenic variantthereof, or an immunogenic fragment of the SARS-CoV-2 S protein or theimmunogenic variant thereof, e.g., as encoded by the RNA describedherein, comprising said mutations comprises the amino acid sequence ofSEQ ID NO: 49, an amino acid sequence having at least 99.5%, 99%, 98.5%,98%, 98.5%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acidsequence of SEQ ID NO: 49, or an immunogenic fragment of the amino acidsequence of SEQ ID NO: 49, or the amino acid sequence having at least99.5%, 99%, 98.5%, 98%, 98.5%, 97%, 96%, 95%, 90%, 85%, or 80% identityto the amino acid sequence of SEQ ID NO: 49. In some embodiments, aSARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenicfragment of the SARS-CoV-2 S protein or the immunogenic variant thereof,e.g., as encoded by the RNA described herein, comprising said mutationscomprises the amino acid sequence of SEQ ID NO: 49.

In some embodiments, a SARS-CoV-2 S protein, an immunogenic variantthereof, or an immunogenic fragment of the SARS-CoV-2 S protein or theimmunogenic variant thereof, e.g., as encoded by the RNA describedherein, comprising said mutations comprises the amino acid sequence ofSEQ ID NO: 52, an amino acid sequence having at least 99.5%, 99%, 98.5%,98%, 98.5%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acidsequence of SEQ ID NO: 52, or an immunogenic fragment of the amino acidsequence of SEQ ID NO: 52, or the amino acid sequence having at least99.5%, 99%, 98.5%, 98%, 98.5%, 97%, 96%, 95%, 90%, 85%, or 80% identityto the amino acid sequence of SEQ ID NO: 52. In some embodiments, aSARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenicfragment of the SARS-CoV-2 S protein or the immunogenic variant thereof,e.g., as encoded by the RNA described herein, comprising said mutationscomprises the amino acid sequence of SEQ ID NO: 52.

In some embodiments, the methods and agents, e.g., RNA (e.g., mRNA)compositions, described herein following administration to a subjectinduce a cell-mediated immune response (e.g., CD4+ and/or CD8+ T cellresponse). In some embodiments, T cells are induced that recognize oneor more epitopes (e.g., MHC class I-restricted epitopes) selected fromthe group consisting of LPFNDGVYF (SEQ ID NO: 145), GVYFASTEK (SEQ IDNO: 146), YLQPRTFLL (SEQ ID NO: 138), QPTESIVRF (SEQ ID NO: 143),CVADYSVLY (SEQ ID NO: 147), KCYGVSPTK (SEQ ID NO: 151), NYNYLYRLF (SEQID NO: 141), FQPTNGVGY (SEQ ID NO: 148), IPFAMQMAY (SEQ ID NO: 144),RLQSLQTYV (SEQ ID NO: 139), GTHWFVTQR (SEQ ID NO: 149), VYDPLQPEL (SEQID NO: 150), QYIKWPWYI (SEQ ID NO: 140), and KWPWYIWLGF (SEQ ID NO:142). In one embodiment, T cells are induced that recognize the epitopeYLQPRTFLL (SEQ ID NO: 138). In one embodiment, T cells are induced thatrecognize the epitope NYNYLYRLF (SEQ ID NO: 141). In one embodiment, Tcells are induced that recognize the epitope QYIKWPWYI (SEQ ID NO: 140).In one embodiment, T cells are induced that recognize the epitopeKCYGVSPTK (SEQ ID NO: 151). In one embodiment, T cells are induced thatrecognize the epitope RLQSLQTYV (SEQ ID NO: 139). In some embodiments,the methods and agents, e.g., RNA (e.g., mRNA) compositions, describedherein are administered according to a regimen which achieves suchinduction of T cells.

In some embodiments, the methods and agents, e.g., RNA (e.g., mRNA)compositions, described herein following administration to a subjectinduce a cell-mediated immune response (e.g., CD4+ and/or CD8+ T cellresponse) that is detectable 15 weeks or later, 16 weeks or later, 17weeks or later, 18 weeks or later, 19 weeks or later, 20 weeks or later,21 weeks or later, 22 weeks or later, 23 weeks or later, 24 weeks orlater or 25 weeks or later after administration, e.g., using two dosesof the RNA described herein (wherein the second dose may be administeredabout 21 days following administration of the first dose). In someembodiments, the methods and agents, e.g., RNA (e.g., mRNA)compositions, described herein are administered according to a regimenwhich achieves such induction of a cell-mediated immune response.

In one embodiment, vaccination against Coronavirus described herein,e.g., using RNA described herein which may be administered in theamounts and regimens described herein, e.g., at two doses of 30 μg perdose e.g. administered 21 days apart, may be repeated after a certainperiod of time, e.g., once it is observed that protection againstCoronavirus infection diminishes, using the same or a different vaccineas used for the first vaccination. Such certain period of time may be atleast 6 months, 1 year, two years etc. In one embodiment, the same RNAas used for the first vaccination is used for the second or furthervaccination, however, at a lower dose or a lower frequency ofadministration. For example, the first vaccination may comprisevaccination using a dose of about 30 μg per dose, wherein in oneembodiment, at least two of such doses are administered, (for example, asecond dose may be administered about 21 days following administrationof the first dose) and the second or further vaccination may comprisevaccination using a dose of less than about 30 μg per dose, wherein inone embodiment, only one of such doses is administered. In oneembodiment, a different RNA as used for the first vaccination is usedfor the second or further vaccination, e.g., BNT162b2 is used for thefirst vaccination and BNT162B1 or BNT162b3 is used for the second orfurther vaccination.

In one embodiment, the vaccination regimen comprises a first vaccinationusing at least two doses of the RNA described herein, e.g., two doses ofthe RNA described herein (wherein the second dose may be administeredabout 21 days following administration of the first dose), and a secondvaccination using a single dose or multiple doses, e.g., two doses, ofthe RNA described herein. In various embodiments, the second vaccinationis administered 3 to 24 months, 6 to 18 months, 6 to 12 months, or 5 to7 months after administration of the first vaccination, e.g., after theinitial two-dose regimen. The amount of RNA used in each dose of thesecond vaccination may be equal or different to the amount of RNA usedin each dose of the first vaccination. In one embodiment, the amount ofRNA used in each dose of the second vaccination is equal to the amountof RNA used in each dose of the first vaccination. In one embodiment,the amount of RNA used in each dose of the second vaccination and theamount of RNA used in each dose of the first vaccination is about 30 μgper dose. In one embodiment, the same RNA as used for the firstvaccination is used for the second vaccination.

In one embodiment, the RNA used for the first vaccination and for thesecond vaccination is BNT162b2.

In some embodiments, when the RNA used for the first vaccination and forthe second vaccination is BNT162b2, the aim is to induce an immuneresponse that targets SARS-CoV-2 variants including, but not limited to,the Omicron (B.1.1.529) variant. Accordingly, in some embodiments, whenthe RNA used for the first vaccination and for the second vaccination isBNT162b2, the aim is to protect a subject from infection with SARS-CoV-2variants including, but not limited to, the Omicron (B.1.1.529) variant.

In one embodiment, a different RNA as used for the first vaccination isused for the second vaccination. In one embodiment, the RNA used for thefirst vaccination is BNT162b2 and the RNA used for the secondvaccination is RNA encoding a SARS-CoV-2 S protein of a SARS-CoV-2variant strain, e.g., a strain discussed herein. In one embodiment, theRNA used for the first vaccination is BNT162b2 and the RNA used for thesecond vaccination is RNA encoding a SARS-CoV-2 S protein of aSARS-CoV-2 variant strain that is prevalent or rapidly spreading at thetime of the second vaccination. In one embodiment, the RNA used for thefirst vaccination is BNT162b2 and the RNA used for the secondvaccination is RNA encoding a SARS-CoV-2 S protein, an immunogenicvariant thereof, or an immunogenic fragment of the SARS-CoV-2 S proteinor the immunogenic variant thereof comprising one or more of themutations described herein for S protein variants such as SARS-CoV-2 Sprotein variants, in particular naturally occurring S protein variants.In one embodiment, the RNA used for the first vaccination is BNT162b2and the RNA used for the second vaccination is RNA encoding a SARS-CoV-2S protein, an immunogenic variant thereof, or an immunogenic fragment ofthe SARS-CoV-2 S protein or the immunogenic variant thereof comprisingone or more mutations, such as all mutations, of a SARS-CoV-2 S proteinof a SARS-CoV-2 variant selected from the group consisting ofVOC-202012/01, 501.V2, Cluster 5, B.1.1.248, and Omicron (B.1.1.529).

In one embodiment, the RNA used for the first vaccination encodes apolypeptide comprising an amino acid sequence with proline residuesubstitutions at positions 986 and 987 of SEQ ID NO:1 and the RNA usedfor the second vaccination is RNA encoding a polypeptide comprising anamino acid sequence with alanine substitution at position 80, glycinesubstitution at position 215, lysine substitution at position 484,tyrosine substitution at position 501, valine substitution at position701, phenylalanine substitution at position 18, isoleucine substitutionat position 246, asparagine substitution at position 417, glycinesubstitution at position 614, deletions at positions 242 to 244, andproline substitutions at positions 986 and 987 of SEQ ID NO:1.

In one embodiment, the RNA used for the first vaccination encodes apolypeptide comprising an amino acid sequence with proline residuesubstitutions at positions 986 and 987 of SEQ ID NO:1 and the RNA usedfor the second vaccination is RNA encoding a polypeptide comprising anamino acid sequence with the following mutations in SEQ ID NO:1:

A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D,S371L, S373P, 5375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R,G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K,D796Y, N856K, Q954H, N969K, L981F, K986P and V987P, as compared to SEQID NO: 1.

In one embodiment, the RNA used for the first vaccination encodes apolypeptide comprising an amino acid sequence with following mutationsin SEQ ID NO: 1: residue substitutions at positions 986 and 987 of SEQID NO:1 and the RNA used for the second vaccination is RNA encoding apolypeptide comprising an amino acid sequence with the followingmutations in SEQ ID NO:1:

T19I, Δ24-26, A27S, G142D, V213G, G339D, S371F, S373P, S375F, T376A,D405N, R408S, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y,Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, K986P,and V987P, as compared to SEQ ID NO: 1.

In one embodiment, the RNA used for the first vaccination encodes apolypeptide comprising an amino acid sequence with proline residuesubstitutions at positions 986 and 987 of SEQ ID NO:1 and the RNA usedfor the second vaccination is RNA encoding a polypeptide comprising anamino acid sequence with the following mutations in SEQ ID NO:1:

T19I, Δ24-26, A27S, Δ69/70, G142D, V213G, G339D, S371F, S373P, S375F,T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V,Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H,N969K, K986P, and V987P, as compared to SEQ ID NO: 1.

In one embodiment, the RNA used for the first vaccination encodes apolypeptide comprising an amino acid sequence with following mutationsin SEQ ID NO: 1:

A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D,S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R,G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K,D796Y, N856K, Q954H, N969K, L981F, K986P and V987P, and the RNA used forthe second vaccination encodes a polypeptide comprising an amino acidsequence with the following mutations in SEQ ID NO: 1:T19I, Δ24-26, A27S, G142D, V213G, G339D, S371F, S373P, S375F, T376A,D405N, R408S, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y,Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, K986P,and V987P, as compared to SEQ ID NO: 1.

In one embodiment, the RNA used for the first vaccination encodes apolypeptide comprising an amino acid sequence with following mutationsin SEQ ID NO: 1:

A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D,S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R,G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K,D796Y, N856K, Q954H, N969K, L981F, K986P and V987P, and the RNA used forthe second vaccination encodes a polypeptide comprising an amino acidsequence with the following mutations in SEQ ID NO: 1:T19I, Δ24-26, A27S, Δ69/70, G142D, V213G, G339D, S371F, S373P, S375F,T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V,Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H,N969K, K986P, and V987P, as compared to SEQ ID NO: 1.

In one embodiment, the RNA used for the first vaccination encodes apolypeptide comprising an amino acid sequence with following mutationsin SEQ ID NO: 1:

T19I, Δ24-26, A27S, G142D, V213G, G339D, S371F, S373P, S375F, T376A,D405N, R408S, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y,Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, K986P,and V987P, as compared to SEQ ID NO: 1, and the RNA used for the secondvaccination encodes a polypeptide comprising an amino acid sequence withthe following mutations in SEQ ID NO: 1:T19I, Δ24-26, A27S, Δ69/70, G142D, V213G, G339D, S371F, S373P, S375F,T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V,Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H,N969K, K986P, and V987P, as compared to SEQ ID NO: 1.

In one embodiment, the RNA used for the first vaccination encodes apolypeptide comprising the amino acid sequence of SEQ ID NO: 7 and theRNA used for the second vaccination is RNA encoding a polypeptidecomprising the amino acid sequence of SEQ ID NO: 49.

In one embodiment, the RNA used for the first vaccination comprises thenucleotide sequence of SEQ ID NO: 20 and the RNA used for the secondvaccination is RNA comprising the nucleotide sequence of SEQ ID NO: 51.

In one embodiment, the RNA used for the first vaccination encodes apolypeptide comprising the amino acid sequence of SEQ ID NO: 7 and theRNA used for the second vaccination is RNA encoding a polypeptidecomprising the amino acid sequence of SEQ ID NO: 55, 58 or 61.

In one embodiment, the RNA used for the first vaccination comprises thenucleotide sequence of SEQ ID NO: 20 and the RNA used for the secondvaccination is RNA comprising the nucleotide sequence of SEQ ID NO: 57,60, or 63a.

In one embodiment, the RNA used for the first vaccination encodes apolypeptide comprising the amino acid sequence of SEQ ID NO: 58 and theRNA used for the second vaccination is RNA encoding a polypeptidecomprising the amino acid sequence of SEQ ID NO: 49, 55 or 61.

In one embodiment, the RNA used for the first vaccination comprises thenucleotide sequence of SEQ ID NO: 60 and the RNA used for the secondvaccination is RNA comprising the nucleotide sequence of SEQ ID NO: 51,57, or 63a.

In one embodiment, the RNA used for the first vaccination encodes apolypeptide comprising the amino acid sequence of SEQ ID NO: 49 and theRNA used for the second vaccination is RNA encoding a polypeptidecomprising the amino acid sequence of SEQ ID NO: 55 or 61.

In one embodiment, the RNA used for the first vaccination comprises thenucleotide sequence of SEQ ID NO: 51 and the RNA used for the secondvaccination is RNA comprising the nucleotide sequence of SEQ ID NO: 57or 63a.

In one embodiment, the RNA used for the first vaccination encodes apolypeptide comprising the amino acid sequence of SEQ ID NO: 55 and theRNA used for the second vaccination is RNA encoding a polypeptidecomprising the amino acid sequence of SEQ ID NO: 61.

In one embodiment, the RNA used for the first vaccination comprises thenucleotide sequence of SEQ ID NO: 57 and the RNA used for the secondvaccination is RNA comprising the nucleotide sequence of SEQ ID NO: 63a.

In one embodiment, the RNA used for the first vaccination encodes apolypeptide comprising an amino acid sequence with proline residuesubstitutions at positions 986 and 987 of SEQ ID NO:1 and the RNA usedfor the second vaccination is RNA encoding a polypeptide comprising anamino acid sequence with the following mutations in SEQ ID NO:1:

A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D,S371L, S373P, S375F, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y,Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H,N969K, L981F, K986P and V987P, as compared to SEQ ID NO: 1. In someembodiments, the polypeptide encoded by the RNA used in the secondvaccination further comprises proline residue substitutions at positionscorresponding to 986 and 987 of SEQ ID NO:1.

In one embodiment, the RNA used for the first vaccination encodes apolypeptide comprising the amino acid sequence of SEQ ID NO: 7 and theRNA used for the second vaccination is RNA encoding a polypeptidecomprising the amino acid sequence of SEQ ID NO: 52.

In one embodiment, a subject is administered a primary regimencomprising two 30 ug doses of RNA comprising a sequence encoding apolypeptide comprising the amino acid sequence of SEQ ID NO: 7, and abooster regimen comprising at least one dose of 30 ug of RNA comprisinga sequence encoding a polypeptide comprising the amino acid sequence ofSEQ ID NO: 7, wherein the booster regimen is administered at least 2months (e.g., at least 3 months, at least 4 months, at least 5 months,or at least 6 months) after administration of the primary regimen, andwherein the subject has optionally previously been administered a firstbooster regimen comprising a 30 ug dose of RNA comprising a sequenceencoding a polypeptide comprising the amino acid sequence of SEQ ID NO:7.

In one embodiment, a subject is administered a primary regimencomprising two 30 ug doses of RNA comprising a sequence encoding apolypeptide comprising the amino acid sequence of SEQ ID NO: 7, and abooster regimen comprising at least one dose of 50 ug of RNA comprisinga sequence encoding a polypeptide comprising the amino acid sequence ofSEQ ID NO: 7, wherein the booster regimen is administered at least 2months (e.g., at least 3 months, at least 4 months, at least 5 months,or at least 6 months) after administration of the primary regimen, andwherein the subject has optionally previously been administered a firstbooster regimen comprising a 30 ug dose of RNA comprising a sequenceencoding a polypeptide comprising the amino acid sequence of SEQ ID NO:7.

In one embodiment, a subject is administered a primary regimencomprising two 30 ug doses of RNA comprising a sequence encoding apolypeptide comprising the amino acid sequence of SEQ ID NO: 7, and abooster regimen comprising at least one dose of 60 ug of RNA comprisinga sequence encoding a polypeptide comprising the amino acid sequence ofSEQ ID NO: 7, wherein the booster regimen is administered at least 2months (e.g., at least 3 months, at least 4 months, at least 5 months,or at least 6 months) after administration of the primary regimen, andwherein the subject has optionally previously been administered a firstbooster regimen comprising a 30 ug dose of RNA comprising a sequenceencoding a polypeptide comprising the amino acid sequence of SEQ ID NO:7.

In one embodiment, a subject is administered a primary regimencomprising two 30 ug doses of RNA comprising a sequence encoding apolypeptide comprising the amino acid sequence of SEQ ID NO: 7, and abooster regimen comprising at least one dose of 30 ug of RNA comprisinga sequence encoding a polypeptide comprising the amino acid sequence ofSEQ ID NO: 49, wherein the booster regimen is administered at least 2months (e.g., at least 3 months, at least 4 months, at least 5 months,or at least 6 months) after administration of the primary regimen, andwherein the subject has optionally previously been administered a firstbooster regimen comprising a 30 ug dose of RNA comprising a sequenceencoding a polypeptide comprising the amino acid sequence of SEQ ID NO:7.

In one embodiment, a subject is administered a primary regimencomprising at least two 30 ug doses of RNA comprising a sequenceencoding a polypeptide comprising the amino acid sequence of SEQ ID NO:7, and a booster regimen comprising at least two doses of 30 ug of RNAcomprising a sequence encoding a polypeptide comprising the amino acidsequence of SEQ ID NO: 49, wherein in some embodiments the two doses ofthe booster regimen are administered at least 2 months apart from eachother (e.g., at least 3 months, at least 4 months, at least 5 months, orat least 6 months apart from each other). In some embodiments, such asubject may have previously been administered a 30 ug dose of RNAcomprising a sequence encoding a polypeptide comprising the amino acidsequence of SEQ ID NO: 7 as a booster dose.

In one embodiment, a subject is administered a primary regimencomprising two 30 ug doses of RNA comprising a sequence encoding apolypeptide comprising the amino acid sequence of SEQ ID NO: 7, and abooster regimen comprising at least one dose of 50 ug of RNA comprisinga nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:49, wherein the booster regimen is administered at least 2 months (e.g.,at least 3 months, at least 4 months, at least 5 months, or at least 6months) after administration of the primary regimen, and wherein thesubject has optionally previously been administered a first boosterregimen comprising a 30 ug dose of RNA comprising a sequence encoding apolypeptide comprising the amino acid sequence of SEQ ID NO: 7.

In one embodiment, a subject is administered a primary regimencomprising two 30 ug doses of RNA comprising a sequence encoding apolypeptide comprising the amino acid sequence of SEQ ID NO: 7, and abooster regimen comprising at least one dose of 60 ug of RNA comprisinga nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:49, wherein the booster regimen is administered at least 2 months (e.g.,at least 3 months, at least 4 months, at least 5 months, or at least 6months) after administration of the primary regimen, and wherein thesubject has optionally previously been administered a first boosterregimen comprising a 30 ug dose of RNA comprising a sequence encoding apolypeptide comprising the amino acid sequence of SEQ ID NO: 7.

In some embodiments, a subject is administered a primary regimencomprising two doses of ug of RNA (administered, e.g., about 21 daysafter one another), wherein each 30 ug dose of RNA comprises 15 ug ofRNA comprising a nucleotide sequence encoding the amino acid sequence ofSEQ ID NO: 7 and 15 ug of RNA comprising a nucleotide sequence encodingthe amino acid sequence of SEQ ID NO: 49. In some embodiments, such aprimary regimen is administered to a vaccine naïve subject.

In some embodiments, a subject is administered a primary regimencomprising two doses of ug of RNA (administered, e.g., about 21 daysafter one another), wherein each 30 ug dose of RNA comprises anucleotide sequence encoding the amino acid sequence of SEQ ID NO: 49.In some embodiments, such a primary regimen is administered to a vaccinenaïve subject.

In one embodiment, a subject is administered a primary regimencomprising two 30 ug doses of RNA comprising a sequence encoding apolypeptide comprising the amino acid sequence of SEQ ID NO: 7, and abooster regimen comprising at least one dose of 30 ug of RNA, whereinthe 30 ug of RNA comprises 15 ug of RNA comprising a nucleotide sequenceencoding the amino acid sequence of SEQ ID NO: 7 and 15 ug of RNAcomprising a nucleotide sequence encoding the amino acid sequence of SEQID NO: 49, wherein the two RNAs are optionally administered in the samecomposition, and wherein the booster regimen is administered at least 2months (e.g., at least 3 months, at least 4 months, at least 5 months,or at least 6 months) after administration of the primary regimen, andwherein the subject has optionally previously been administered a firstbooster regimen comprising a 30 ug dose of RNA comprising a sequenceencoding a polypeptide comprising the amino acid sequence of SEQ ID NO:7.

In one embodiment, a subject is administered a primary regimencomprising two 30 ug doses of RNA comprising a sequence encoding apolypeptide comprising the amino acid sequence of SEQ ID NO: 7, and abooster regimen comprising at least one dose of 50 ug of RNA, whereinthe 50 ug of RNA comprises 25 ug of RNA comprising a nucleotide sequenceencoding the amino acid sequence of SEQ ID NO: 7 and 25 ug of RNAcomprising a nucleotide sequence encoding the amino acid sequence of SEQID NO: 49, wherein the two RNAs are optionally administered in the samecomposition (e.g., a formulation comprising both RNAs), and wherein thebooster regimen is administered at least 2 months (e.g., at least 3months, at least 4 months, at least 5 months, or at least 6 months)after administration of the primary regimen, and wherein the subject hasoptionally previously been administered a first booster regimencomprising a 30 ug dose of RNA comprising a sequence encoding apolypeptide comprising the amino acid sequence of SEQ ID NO: 7.

In one embodiment, a subject is administered a primary regimencomprising two 30 ug doses of RNA comprising a sequence encoding apolypeptide comprising the amino acid sequence of SEQ ID NO: 7, and abooster regimen comprising at least one dose of 60 ug of RNA, whereinthe 60 ug of RNA comprises 30 ug of RNA comprising a nucleotide sequenceencoding the amino acid sequence of SEQ ID NO: 7 and 30 ug of an RNAcomprising a nucleotide sequence encoding the amino acid sequence of SEQID NO: 49, wherein the two RNAs are optionally administered in the samecomposition, and wherein the booster regimen is administered at least 2months (e.g., at least 3 months, at least 4 months, at least 5 months,or at least 6 months) after administration of the primary regimen, andwherein the subject has optionally previously been administered a firstbooster regimen comprising a 30 ug dose of RNA comprising a sequenceencoding a polypeptide comprising the amino acid sequence of SEQ ID NO:7.

In one embodiment, the RNA used for the first vaccination comprises thenucleotide sequence of SEQ ID NO: 20 and the RNA used for the secondvaccination is RNA comprising the nucleotide sequence of SEQ ID NO: 54.

In one embodiment, a subject is administered a primary regimencomprising two 30 ug doses of RNA comprising a nucleotide sequence ofSEQ ID NO: 20, and a booster regimen comprising at least one dose of 30ug of RNA comprising a nucleotide sequence of SEQ ID NO: 20, wherein thebooster regimen is administered at least 2 months (e.g., at least 3months, at least 4 months, at least 5 months, or at least 6 months)after administration of the primary regimen, and wherein the subject hasoptionally previously been administered a first booster regimencomprising a 30 ug dose of RNA comprising a nucleotide sequence of SEQID NO: 20.

In one embodiment, a subject is administered a primary regimencomprising two 30 ug doses of RNA comprising a nucleotide sequence ofSEQ ID NO: 20, and a booster regimen comprising at least one dose of 50ug of RNA comprising a nucleotide sequence of SEQ ID NO: 20, wherein thebooster regimen is administered at least 2 months (e.g., at least 3months, at least 4 months, at least 5 months, or at least 6 months)after administration of the primary regimen, and wherein the subject hasoptionally previously been administered a first booster regimencomprising a 30 ug dose of RNA comprising a nucleotide sequence of SEQID NO: 20.

In one embodiment, a subject is administered a primary regimencomprising two 30 ug doses of RNA comprising a nucleotide sequence ofSEQ ID NO: 20, and a booster regimen comprising at least one dose of 60ug of RNA comprising a nucleotide sequence of SEQ ID NO: 20, wherein thebooster regimen is administered at least 2 months (e.g., at least 3months, at least 4 months, at least 5 months, or at least 6 months)after administration of the primary regimen, and wherein the subject hasoptionally previously been administered a first booster regimencomprising a 30 ug dose of RNA comprising a nucleotide sequence of SEQID NO: 20.

In one embodiment, a subject is administered a primary regimencomprising two 30 ug doses of RNA comprising a nucleotide sequence ofSEQ ID NO: 20, and a booster regimen comprising at least one dose of 30ug of RNA comprising a nucleotide sequence of SEQ ID NO: 51, wherein thebooster regimen is administered at least 2 months (e.g., at least 3months, at least 4 months, at least 5 months, or at least 6 months)after administration of the primary regimen, and wherein the subject hasoptionally previously been administered a first booster regimencomprising a 30 ug dose of RNA comprising a nucleotide sequence of SEQID NO: 20.

In one embodiment, a subject is administered a primary regimencomprising two 30 ug doses of RNA comprising a nucleotide sequence ofSEQ ID NO: 20, and a booster regimen comprising at least one dose of 50ug of RNA comprising a nucleotide sequence of SEQ ID NO: 51, wherein thebooster regimen is administered at least 2 months (e.g., at least 3months, at least 4 months, at least 5 months, or at least 6 months)after administration of the primary regimen, and wherein the subject hasoptionally previously been administered a first booster regimencomprising a 30 ug dose of RNA comprising a nucleotide sequence of SEQID NO: 20.

In one embodiment, a subject is administered a primary regimencomprising two 30 ug doses of RNA comprising a nucleotide sequence ofSEQ ID NO: 20, and a booster regimen comprising at least one dose of 60ug of RNA comprising a nucleotide sequence of SEQ ID NO: 51, wherein thebooster regimen is administered at least 2 months (e.g., at least 3months, at least 4 months, at least 5 months, or at least 6 months)after administration of the primary regimen, and wherein the subject hasoptionally previously been administered a first booster regimencomprising a 30 ug dose of RNA comprising a nucleotide sequence of SEQID NO: 20.

In one embodiment, a subject is administered a primary regimencomprising two 30 ug doses of RNA comprising a nucleotide sequence ofSEQ ID NO: 20, and a booster regimen comprising at least one dose of 30ug of RNA, wherein the 30 ug of RNA comprises 15 ug of RNA comprising anucleotide sequence of SEQ ID NO: 20 and 15 ug of RNA comprising anucleotide sequence of SEQ ID NO: 51, wherein the two RNAs areoptionally administered in the same composition, and wherein the boosterregimen is administered at least 2 months (e.g., at least 3 months, atleast 4 months, at least 5 months, or at least 6 months) afteradministration of the primary regimen, and wherein the subject hasoptionally previously been administered a first booster regimencomprising a 30 ug dose of RNA comprising a nucleotide sequence of SEQID NO: 20. In one embodiment, a subject is administered a primaryregimen comprising two 30 ug doses of RNA comprising a nucleotidesequence of SEQ ID NO: 20, and a booster regimen comprising at least onedose comprising 50 ug of a RNA, wherein the 50 ug of RNA comprises 25 ugof RNA comprising a nucleotide sequence of SEQ ID NO: 20 and 25 ug ofRNA comprising a nucleotide sequence of SEQ ID NO: 51, wherein the twoRNAs are optionally administered in the same composition, and whereinthe booster regimen is administered at least 2 months (e.g., at least 3months, at least 4 months, at least 5 months, or at least 6 months)after administration of the primary regimen, and wherein the subject hasoptionally previously been administered a first booster regimencomprising a 30 ug dose of RNA comprising a nucleotide sequence of SEQID NO: 20.

In one embodiment, a subject is administered a primary regimencomprising two 30 ug doses of RNA comprising a nucleotide sequence ofSEQ ID NO: 20, and a booster regimen comprising at least one dosecomprising 60 ug of RNA, wherein the 60 ug of RNA comprises 30 ug of anRNA comprising a nucleotide sequence of SEQ ID NO: 20 and 30 ug of anRNA comprising a nucleotide sequence of SEQ ID NO: 51, wherein the twoRNAs are optionally administered in the same composition, and whereinthe booster regimen is administered at least 2 months (e.g., at least 3months, at least 4 months, at least 5 months, or at least 6 months)after administration of the primary regimen, and wherein the subject hasoptionally previously been administered a first booster regimencomprising a 30 ug dose of RNA comprising a nucleotide sequence of SEQID NO: 20.

In one embodiment, a subject is administered a primary regimencomprising two 30 ug doses of RNA comprising a nucleotide sequence ofSEQ ID NO: 20, and a booster regimen comprising at least one dose of 30ug of RNA comprising a nucleotide sequence of SEQ ID NO: 57, wherein thebooster regimen is administered at least 2 months (e.g., at least 3months, at least 4 months, at least 5 months, or at least 6 months)after administration of the primary regimen, and wherein the subject hasoptionally previously been administered a first booster regimencomprising a 30 ug dose of RNA comprising a nucleotide sequence of SEQID NO: 20.

In one embodiment, a subject is administered a primary regimencomprising two 30 ug doses of RNA comprising a nucleotide sequence ofSEQ ID NO: 20, and a booster regimen comprising at least one dose of 50ug of RNA comprising a nucleotide sequence of SEQ ID NO: 57, wherein thebooster regimen is administered at least 2 months (e.g., at least 3months, at least 4 months, at least 5 months, or at least 6 months)after administration of the primary regimen, and wherein the subject hasoptionally previously been administered a first booster regimencomprising a 30 ug dose of RNA comprising a nucleotide sequence of SEQID NO: 20.

In one embodiment, a subject is administered a primary regimencomprising two 30 ug doses of RNA comprising a nucleotide sequence ofSEQ ID NO: 20, and a booster regimen comprising at least one dose of 60ug of RNA comprising a nucleotide sequence of SEQ ID NO: 57, wherein thebooster regimen is administered at least 2 months (e.g., at least 3months, at least 4 months, at least 5 months, or at least 6 months)after administration of the primary regimen, and wherein the subject hasoptionally previously been administered a first booster regimencomprising a 30 ug dose of RNA comprising a nucleotide sequence of SEQID NO: 20.

In one embodiment, a subject is administered a primary regimencomprising two 30 ug doses of RNA comprising a nucleotide sequence ofSEQ ID NO: 20, and a booster regimen comprising at least one dose of 30ug of RNA comprising a nucleotide sequence of SEQ ID NO: 60, wherein thebooster regimen is administered at least 2 months (e.g., at least 3months, at least 4 months, at least 5 months, or at least 6 months)after administration of the primary regimen, and wherein the subject hasoptionally previously been administered a first booster regimencomprising a 30 ug dose of RNA comprising a nucleotide sequence of SEQID NO: 20.

In one embodiment, a subject is administered a primary regimencomprising two 30 ug doses of RNA comprising a nucleotide sequence ofSEQ ID NO: 20, and a booster regimen comprising at least one dose of 50ug of RNA comprising a nucleotide sequence of SEQ ID NO: 60, wherein thebooster regimen is administered at least 2 months (e.g., at least 3months, at least 4 months, at least 5 months, or at least 6 months)after administration of the primary regimen, and wherein the subject hasoptionally previously been administered a first booster regimencomprising a 30 ug dose of RNA comprising a nucleotide sequence of SEQID NO: 20.

In one embodiment, a subject is administered a primary regimencomprising two 30 ug doses of RNA comprising a nucleotide sequence ofSEQ ID NO: 20, and a booster regimen comprising at least one dose of 60ug of RNA comprising a nucleotide sequence of SEQ ID NO: 60, wherein thebooster regimen is administered at least 2 months (e.g., at least 3months, at least 4 months, at least 5 months, or at least 6 months)after administration of the primary regimen, and wherein the subject hasoptionally previously been administered a first booster regimencomprising a 30 ug dose of RNA comprising a nucleotide sequence of SEQID NO: 20.

In one embodiment, a subject is administered a primary regimencomprising two 30 ug doses of RNA comprising a nucleotide sequence ofSEQ ID NO: 20, and a booster regimen comprising at least one dose of 30ug of RNA comprising a nucleotide sequence of SEQ ID NO: 63a, whereinthe booster regimen is administered at least 2 months (e.g., at least 3months, at least 4 months, at least 5 months, or at least 6 months)after administration of the primary regimen, and wherein the subject hasoptionally previously been administered a first booster regimencomprising a 30 ug dose of RNA comprising a nucleotide sequence of SEQID NO: 20.

In one embodiment, a subject is administered a primary regimencomprising two 30 ug doses of RNA comprising a nucleotide sequence ofSEQ ID NO: 20, and a booster regimen comprising at least one dose of 50ug of RNA comprising a nucleotide sequence of SEQ ID NO: 63a, whereinthe booster regimen is administered at least 2 months (e.g., at least 3months, at least 4 months, at least 5 months, or at least 6 months)after administration of the primary regimen, and wherein the subject hasoptionally previously been administered a first booster regimencomprising a 30 ug dose of RNA comprising a nucleotide sequence of SEQID NO: 20.

In one embodiment, a subject is administered a primary regimencomprising two 30 ug doses of RNA comprising a nucleotide sequence ofSEQ ID NO: 63a, and a booster regimen comprising at least one dose of 60ug of RNA comprising a nucleotide sequence of SEQ ID NO: 57, wherein thebooster regimen is administered at least 2 months (e.g., at least 3months, at least 4 months, at least 5 months, or at least 6 months)after administration of the primary regimen, and wherein the subject hasoptionally previously been administered a first booster regimencomprising a 30 ug dose of RNA comprising a nucleotide sequence of SEQID NO: 20. In one embodiment, the vaccination regimen comprises a firstvaccination using two doses of RNA encoding a polypeptide comprising anamino acid sequence with proline residue substitutions at positions 986and 987 of SEQ ID NO:1 administered about 21 days apart and a secondvaccination using a single dose or multiple doses of RNA encoding apolypeptide comprising an amino acid sequence with proline residuesubstitutions at positions 986 and 987 of SEQ ID NO:1 administered about4 to 12 months, 5 to 12 months, or 6 to 12 months after administrationof the first vaccination, i.e., after the initial two-dose regimen. Inone embodiment, each RNA dose comprises 30 μg RNA. In this embodiment,the aim in one embodiment is to induce an immune response that targetsSARS-CoV-2 variants including, but not limited to, the Omicron(B.1.1.529) variant. Accordingly, in this embodiment, the aim in oneembodiment is to protect a subject from infection with SARS-CoV-2variants including, but not limited to, the Omicron (B.1.1.529) variant.

In one embodiment, the vaccination regimen comprises a first vaccinationusing two doses of RNA encoding a polypeptide comprising an amino acidsequence with proline residue substitutions at positions 986 and 987 ofSEQ ID NO:1 administered about 21 days apart and a second vaccinationusing a single dose or multiple doses of RNA encoding a polypeptidecomprising an amino acid sequence with alanine substitution at position80, glycine substitution at position 215, lysine substitution atposition 484, tyrosine substitution at position 501, valine substitutionat position 701, phenylalanine substitution at position 18, isoleucinesubstitution at position 246, asparagine substitution at position 417,glycine substitution at position 614, deletions at positions 242 to 244,and proline substitutions at positions 986 and 987 of SEQ ID NO:1administered about 6 to 12 months after administration of the firstvaccination, i.e., after the initial two-dose regimen. In oneembodiment, each RNA dose comprises 30 μg RNA.

In one embodiment, the vaccination regimen comprises a first vaccinationusing two doses of RNA encoding a polypeptide comprising an amino acidsequence with proline residue substitutions at positions 986 and 987 ofSEQ ID NO:1 administered about 21 days apart and a second vaccinationusing a single dose or multiple doses of RNA encoding a polypeptidecomprising an amino acid sequence with the following mutations in SEQ IDNO:1:

A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D,S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R,G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K,D796Y, N856K, Q954H, N969K, L981F, K986P and V987P, as compared to SEQID NO: 1, administered after, e.g., about 6 to 12 months afteradministration of the first vaccination, i.e., after the initialtwo-dose regimen. In one embodiment, each RNA dose comprises 30 μg RNA.

In one embodiment, the vaccination regimen comprises a first vaccinationusing two doses of RNA encoding a polypeptide comprising an amino acidsequence with proline residue substitutions at positions 986 and 987 ofSEQ ID NO:1 administered about 21 days apart and a second vaccinationusing a single dose or multiple doses of RNA encoding a polypeptidecomprising an amino acid sequence with the following mutations in SEQ IDNO:1:

A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D,S371L, S373P, S375F, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y,Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H,N969K, L981F, K986P and V987P, as compared to SEQ ID NO: 1, administeredafter, e.g., about 6 to 12 months after administration of the firstvaccination, i.e., after the initial two-dose regimen. In oneembodiment, each RNA dose comprises 30 μg RNA. In some embodiments, theencoded polypeptide further comprises proline residue substitutions atpositions corresponding to 986 and 987 of SEQ ID NO:1.

In one embodiment, the vaccination regimen comprises a first vaccinationinvolving at least two doses of RNA encoding a polypeptide comprising anamino acid sequence with proline residue substitutions at positions 986and 987 of SEQ ID NO: 1 administered about 21 days apart and a secondvaccination involving a single dose or multiple doses of RNA encoding apolypeptide comprising an amino acid sequence with the followingmutations in SEQ ID NO: 1:

T19I, Δ24-26, A27S, G142D, V213G, G339D, S371F, 5373P, S375F, T376A,D405N, R408S, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y,Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, K986P,and V987P, as compared to SEQ ID NO: 1, administered after, e.g., about6 to 12 months after administration of the first vaccination, i.e.,after the initial two-dose regimen. In one embodiment, each or at leastone RNA dose comprises 30 ug RNA.

In one embodiment, the vaccination regimen comprises a first vaccinationinvolving at least two doses of RNA encoding a polypeptide comprising anamino acid sequence with proline residue substitutions at positions 986and 987 of SEQ ID NO:1 administered about 21 days apart and a secondvaccination involving a single dose or multiple doses of RNA encoding apolypeptide comprising an amino acid sequence with the followingmutations in SEQ ID NO:1:

T19I, Δ24-26, A27S, Δ69/70, G142D, V213G, G339D, S371F, S373P, S375F,T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V,Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H,N969K, K986P, and V987P, as compared to SEQ ID NO: 1 administered after,e.g., about 6 to 12 months after administration of the firstvaccination, i.e., after the initial two-dose regimen. In oneembodiment, each or at least one RNA dose comprises 30 μg RNA.

In one embodiment, the vaccination regimen comprises a first vaccinationinvolving at least two doses of RNA encoding a polypeptide comprising anamino acid sequence with the following mutations in SEQ ID NO:1:

A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D,S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R,G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K,D796Y, N856K, Q954H, N969K, L981F, K986P and V987P, wherein the twodoses of the first vaccination are administered about 21 days apart andwherein the vaccination regimen comprises a second vaccination involvinga single dose or multiple doses of RNA encoding a polypeptide comprisingan amino acid sequence with the following mutations in SEQ ID NO:1:T19I, Δ24-26, A27S, G142D, V213G, G339D, S371F, S373P, S375F, T376A,D405N, R408S, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y,Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, K986P,and V987P, as compared to SEQ ID NO: 1 administered after, e.g., about 6to 12 months after administration of the first vaccination, i.e., afterthe initial two-dose regimen. In one embodiment, each or at least oneRNA dose comprises 30 μg RNA.

In one embodiment, the vaccination regimen comprises a first vaccinationinvolving at least two doses of RNA encoding a polypeptide comprising anamino acid sequence with the following mutations in SEQ ID NO:1:

A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D,S371L, S373P, 5375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R,G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K,D796Y, N856K, Q954H, N969K, L981F, K986P and V987P, wherein the twodoses of the first vaccination are administered about 21 days apart andwherein the vaccination regimen comprises a second vaccination involvinga single dose or multiple doses of RNA encoding a polypeptide comprisingan amino acid sequence with the following mutations in SEQ ID NO:1:T19I, Δ24-26, A27S, Δ69/70, G142D, V213G, G339D, S371F, S373P, S375F,T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V,Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H,N969K, K986P, and V987P, as compared to SEQ ID NO: 1 administered after,e.g., about 6 to 12 months after administration of the firstvaccination, i.e., after the initial two-dose regimen. In oneembodiment, each or at least one RNA dose comprises 30 μg RNA.

In one embodiment, the vaccination regimen comprises a first vaccinationinvolving at least two doses of RNA encoding a polypeptide comprising anamino acid sequence with the following mutations in SEQ ID NO:1:

T19I, Δ24-26, A27S, G142D, V213G, G339D, S371F, S373P, S375F, T376A,D405N, R408S, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y,Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, K986P,and V987P, as compared to SEQ ID NO: 1, wherein the two doses of thefirst vaccination are administered about 21 days apart and wherein thevaccination regimen comprises a second vaccination involving a singledose or multiple doses of RNA encoding a polypeptide comprising an aminoacid sequence with the following mutations in SEQ ID NO:1:T19I, Δ24-26, A27S, Δ69/70, G142D, V213G, G339D, S371F, S373P, S375F,T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V,Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H,N969K, K986P, and V987P, as compared to SEQ ID NO: 1 administered after,e.g., about 6 to 12 months after administration of the firstvaccination, i.e., after the initial two-dose regimen. In oneembodiment, each or at least one RNA dose comprises 30 μg RNA.

In one embodiment, a vaccination regimen comprises (i) a firstvaccination comprising at least three doses of an RNA described herein(e.g., where each dose comprises about 30 ug of an RNA comprising anucleotide sequence of SEQ ID NO: 20), wherein a second dose may beadministered about 21 days following administration of a first dose, anda third dose may be administered at least about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, or 12 months after a second dose; and (ii) a secondvaccination comprising at least one dose of an RNA described herein(e.g., wherein each dose comprises about 30 μg RNA per dose). In someembodiments, a second vaccination comprises at least one dose of abivalent vaccine described herein, e.g., about 30 μg total of a bivalentvaccine, e.g., a bivalent vaccine comprising about 15 μg RNA encoding aSARS-CoV-2 S protein from a Wuhan strain and about 15 μg RNA encoding aSARS-CoV-2 S protein comprising mutations characteristic of an Omicronvariant (b2+Omi). In some embodiments, the bivalent vaccine comprisesabout 15 μg RNA encoding a SARS-CoV-2 S protein from a Wuhan strain andabout 15 μg RNA encoding a SARS-CoV-2 S protein comprising mutationscharacteristic of a BA.1 Omicron variant (e.g., 15 μg RNA comprising asequence of SEQ ID NO: 20 and 15 μg of RNA comprising a sequence of SEQID NO: 51). In some embodiments, the bivalent vaccine comprises about 15μg RNA encoding a SARS-CoV-2 S protein from a Wuhan strain and about 15μg RNA encoding a SARS-CoV-2 S protein comprising mutationscharacteristic of a BA.4/5 Omicron variant (e.g., 15 μg RNA comprising asequence of SEQ ID NO: 20 and 15 μg of RNA comprising a sequence of SEQID NO: 72). In some embodiments, the vaccination regimen is administeredto a subject who is at least about 12 years old. In some embodiments,the vaccination regimen is administered to a subject who is at leastabout 6 months old to less than about 12 years old.

In one embodiment, the second vaccination results in a boosting of theimmune response.

In one embodiment, RNA described herein is co-administered with othervaccines. In some embodiments, RNA described herein is co-administeredwith a composition comprising one or more T-cell epitopes of SARS-CoV-2or RNA encoding the same. In some embodiments, RNA described herein isco-administered one or more T-cell epitopes, or RNA encoding the same,derived from an M protein, an N protein, and/or an ORF1ab protein ofSARS-CoV-2, e.g., a composition disclosed in WO2021188969, the contentsof which is incorporated by reference herein in its entirety. In someembodiments, RNA described herein (e.g., RNA encoding a SARS-CoV-2 Sprotein comprising mutations characteristic of a BA.1, BA.2, or BA.4/5Omicron variant, optionally administered with RNA encoding a SARS-CoV-2S protein of a Wuhan variant) is co-administered with a T-stringconstruct described in WO2021188969 (e.g., an RNA encoding SEQ ID NO: RSC7p2full of WO2021/188969). In some embodiments, RNA described hereinand a T-string construct described in WO2021188969 are administered in acombination of up to about 100 ug RNA total. In some embodiments,subjects are administered with at least 2 doses of RNA described herein(e.g., in some embodiments at 30 ug each) in combination with a T-stringconstruct (e.g., an RNA encoding SEQ ID NO: RS C7p2full ofWO2021/188969), e.g., each dose of a combination of RNA described hereinand an RNA encoding SEQ ID NO: RS C7p2full of up to about 100 ug RNAtotal, wherein the two doses are administered, for example, at least 4weeks or longer (including, e.g., at least 5 weeks, at least 6 weeks, atleast 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, atleast 11 weeks, or at least 12 weeks, or longer) apart from one another.In some embodiments, subjects are administered at least 3 doses of RNAdescribed herein (e.g., in some embodiments at 30 ug each) incombination with a T-string construct (e.g., an RNA encoding SEQ ID NO:RS C7p2full of WO2021/188969), e.g., each dose of a combination of RNAdescribed herein and an RNA encoding SEQ ID NO: RS C7p2full of up toabout 100 ug RNA total, wherein the first and the second doses and thesecond and third doses are each independently administered at least 4weeks or longer (including, e.g., at least 5 weeks, at least 6 weeks, atleast 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, atleast 11 weeks, or at least 12 weeks, or longer) apart from one another.In some embodiments, the RNA described herein and the T-string constructmay be co-administered as separate formulations (e.g., formulationsadministered on the same day to separate injection sites). In someembodiments, the RNA described herein and the T-string construct may beco-administered as a co-formulation (e.g., a formulation comprising RNAdescribed herein and the T-string construct as separate LNP formulationsor as LNP formulations comprising both a T-string construct and RNAdescribed herein).

In some embodiments, an RNA composition described herein isco-administered with one or more vaccines against a non-SARS-CoV-2disease. In some embodiments, an RNA composition described herein isco-administered with one or more vaccines against a non-SARS-COV-2 viraldisease. In some embodiments, an RNA composition described herein isco-administered with one or more vaccines against a non-SARS-CoV-2respiratory disease. In some embodiments, the non-SARS-CoV-2 respiratorydisease is a non-SARS-CoV-2 Coronavirus, an Influenza virus, aPneumoviridae virus, or a Paramyxoviridae virus. In some embodiments,the Pneumoviridae virus is a Respiratory syncytial virus or aMetapneumovirus. In some embodiments, the Metapneumovirus is a humanmetapneumovirus (hMPV). In some embodiments, the Paramyxoviridae virusis a Parainfluenza virus or a Henipavirus. In some embodiments theparainfluenzavirus is PIV3. In some embodiments, the non-SAR-CoV-2coronavirus is a betacoronavirus (e.g., SARS-CoV-1). In come embodimentsthe non-SARS-CoV-2 coronavirus is a Merbecovirus (e.g., a MERS-CoVvirus).

In some embodiments, an RNA composition described herein isco-administered with an RSV vaccine (e.g., an RSV A or RSV B vaccine).In some embodiments, the RSV vaccine comprises an RSV fusion protein(F), an RSV attachment protein (G), an RSV small hydrophobic protein(SH), an RSV matrix protein (M), an RSV nucleoprotein (N), an RSV M2-1protein, an RSV Large polymerase (L), and/or an RSV phosphoprotein (P),or an immunogenic fragment of immunogenic variant thereof, or a nucleicacid (e.g., RNA), encoding any one of the same.

In some embodiments, an RNA composition described herein isco-administered with an influenza vaccine. In some embodiments, theinfluenza vaccine is an alphainfluenza virus, a betainfluenza virus, agammainfluenza virus or a deltainfluenza virus vaccine. In someembodiments the vaccine is an Influenza A virus, an Influenza B virus,an Influenza C virus, or an Influenza D virus vaccine. In someembodiments, the influenza A virus vaccine comprises a hemagglutininselected from H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13,H14, H15, H16, H17, and H18, or an immunogenic fragment or variant ofthe same, or a nucleic acid (e.g., RNA) encoding any one of the same. Insome embodiments the influenza A vaccine comprises or encodes aneuraminidase (NA) selected from N1, N2, N3, N4, N5, N6, N7, N8, N9,N10, and N11, or an immunogenic fragment or variant of the same, or anucleic acid (e.g., RNA) encoding any one of the same. In someembodiments, the influenza vaccine comprises at least one Influenzavirus hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrixprotein 1 (M1), matrix protein 2 (M2), non-structural protein 1 (NS1),non-structural protein 2 (NS2), nuclear export protein (NEP), polymeraseacidic protein (PA), polymerase basic protein PB1, PB1-F2, and/orpolymerase basic protein 2 (PB2), or an immunogenic fragment or variantthereof, or a nucleic acid (e.g., RNA) encoding any of one of the same.

In some embodiments, an RNA composition provided herein and otherinjectable vaccine(s) are administered at different times. In someembodiments, an RNA composition provided herein is administered at thesame time as other injectable vaccine(s). In some such embodiments, anRNA composition provided herein and at least one another injectablevaccine(s) are administered at different injection sites. In someembodiments, an RNA composition provided herein is not mixed with anyother vaccine in the same syringe. In some embodiments, an RNAcomposition provided herein is not combined with other coronavirusvaccines as part of vaccination against coronavirus, e.g., SARS-CoV-2.

The term “disease” refers to an abnormal condition that affects the bodyof an individual. A disease is often construed as a medical conditionassociated with specific symptoms and signs. A disease may be caused byfactors originally from an external source, such as infectious disease,or it may be caused by internal dysfunctions, such as autoimmunediseases. In humans, “disease” is often used more broadly to refer toany condition that causes pain, dysfunction, distress, social problems,or death to the individual afflicted, or similar problems for those incontact with the individual. In this broader sense, it sometimesincludes injuries, disabilities, disorders, syndromes, infections,isolated symptoms, deviant behaviors, and atypical variations ofstructure and function, while in other contexts and for other purposesthese may be considered distinguishable categories. Diseases usuallyaffect individuals not only physically, but also emotionally, ascontracting and living with many diseases can alter one's perspective onlife, and one's personality.

In the present context, the term “treatment”, “treating” or “therapeuticintervention” relates to the management and care of a subject for thepurpose of combating a condition such as a disease or disorder. The termis intended to include the full spectrum of treatments for a givencondition from which the subject is suffering, such as administration ofthe therapeutically effective compound to alleviate the symptoms orcomplications, to delay the progression of the disease, disorder orcondition, to alleviate or relief the symptoms and complications, and/orto cure or eliminate the disease, disorder or condition as well as toprevent the condition, wherein prevention is to be understood as themanagement and care of an individual for the purpose of combating thedisease, condition or disorder and includes the administration of theactive compounds to prevent the onset of the symptoms or complications.

The term “therapeutic treatment” relates to any treatment which improvesthe health status and/or prolongs (increases) the lifespan of anindividual. Said treatment may eliminate the disease in an individual,arrest or slow the development of a disease in an individual, inhibit orslow the development of a disease in an individual, decrease thefrequency or severity of symptoms in an individual, and/or decrease therecurrence in an individual who currently has or who previously has hada disease.

The terms “prophylactic treatment” or “preventive treatment” relate toany treatment that is intended to prevent a disease from occurring in anindividual. The terms “prophylactic treatment” or “preventive treatment”are used herein interchangeably.

The terms “individual” and “subject” are used herein interchangeably.They refer to a human or another mammal (e.g. mouse, rat, rabbit, dog,cat, cattle, swine, sheep, horse or primate) that can be afflicted withor is susceptible to a disease or disorder but may or may not have thedisease or disorder. In many embodiments, the individual is a humanbeing. Unless otherwise stated, the terms “individual” and “subject” donot denote a particular age, and thus encompass adults, elderlies,children, and newborns. In some embodiments, the term “subject” includeshumans of age of at least 50, at least 55, at least 60, at least 65, atleast 70, or older. In some embodiments, the term “subject” includeshumans of age of at least 65, such as 65 to 80, 65 to 75, or 65 to 70.In embodiments of the present disclosure, the “individual” or “subject”is a “patient”.

The term “patient” means an individual or subject for treatment, inparticular a diseased individual or subject.

In one embodiment of the present disclosure, the aim is to provide animmune response against coronavirus, and to prevent or treat coronavirusinfection.

A pharmaceutical composition comprising RNA encoding a peptide orprotein comprising an epitope may be administered to a subject to elicitan immune response against an antigen comprising said epitope in thesubject which may be therapeutic or partially or fully protective. Aperson skilled in the art will know that one of the principles ofimmunotherapy and vaccination is based on the fact that animmunoprotective reaction to a disease is produced by immunizing asubject with an antigen or an epitope, which is immunologically relevantwith respect to the disease to be treated. Accordingly, pharmaceuticalcompositions described herein are applicable for inducing or enhancingan immune response. Pharmaceutical compositions described herein arethus useful in a prophylactic and/or therapeutic treatment of a diseaseinvolving an antigen or epitope.

As used herein, “immune response” refers to an integrated bodilyresponse to an antigen or a cell expressing an antigen and refers to acellular immune response and/or a humoral immune response. The immunesystem is divided into a more primitive innate immune system, andacquired or adaptive immune system of vertebrates, each of whichcontains humoral and cellular components.

“Cell-mediated immunity”, “cellular immunity”, “cellular immuneresponse”, or similar terms are meant to include a cellular responsedirected to cells characterized by expression of an antigen, inparticular characterized by presentation of an antigen with class I orclass II MHC. The cellular response relates to immune effector cells, inparticular to cells called T cells or T lymphocytes which act as either“helpers” or “killers”. The helper T cells (also termed CD4⁺ T cells)play a central role by regulating the immune response and the killercells (also termed cytotoxic T cells, cytolytic T cells, CD8⁺ T cells orCTLs) kill diseased cells such as virus-infected cells, preventing theproduction of more diseased cells.

An immune effector cell includes any cell which is responsive to vaccineantigen. Such responsiveness includes activation, differentiation,proliferation, survival and/or indication of one or more immune effectorfunctions. The cells include, in particular, cells with lytic potential,in particular lymphoid cells, and are preferably T cells, in particularcytotoxic lymphocytes, preferably selected from cytotoxic T cells,natural killer (NK) cells, and lymphokine-activated killer (LAK) cells.Upon activation, each of these cytotoxic lymphocytes triggers thedestruction of target cells. For example, cytotoxic T cells trigger thedestruction of target cells by either or both of the following means.First, upon activation T cells release cytotoxins such as perforin,granzymes, and granulysin. Perforin and granulysin create pores in thetarget cell, and granzymes enter the cell and trigger a caspase cascadein the cytoplasm that induces apoptosis (programmed cell death) of thecell. Second, apoptosis can be induced via Fas-Fas ligand interactionbetween the T cells and target cells.

The term “effector functions” in the context of the present disclosureincludes any functions mediated by components of the immune system thatresult, for example, in the neutralization of a pathogenic agent such asa virus and/or in the killing of diseased cells such as virus-infectedcells. In one embodiment, the effector functions in the context of thepresent disclosure are T cell mediated effector functions. Suchfunctions comprise in the case of a helper T cell (CD4⁺ T cell) therelease of cytokines and/or the activation of CD8⁺ lymphocytes (CTLs)and/or B cells, and in the case of CTL the elimination of cells, i.e.,cells characterized by expression of an antigen, for example, viaapoptosis or perforin-mediated cell lysis, production of cytokines suchas IFN-γ and TNF-α, and specific cytolytic killing of antigen expressingtarget cells.

The term “immune effector cell” or “immunoreactive cell” in the contextof the present disclosure relates to a cell which exerts effectorfunctions during an immune reaction. An “immune effector cell” in oneembodiment is capable of binding an antigen such as an antigen presentedin the context of MHC on a cell or expressed on the surface of a celland mediating an immune response. For example, immune effector cellscomprise T cells (cytotoxic T cells, helper T cells, tumor infiltratingT cells), B cells, natural killer cells, neutrophils, macrophages, anddendritic cells. Preferably, in the context of the present disclosure,“immune effector cells” are T cells, preferably CD4⁺ and/or CD8⁺ Tcells, most preferably CD8⁺ T cells. According to the presentdisclosure, the term “immune effector cell” also includes a cell whichcan mature into an immune cell (such as T cell, in particular T helpercell, or cytolytic T cell) with suitable stimulation. Immune effectorcells comprise CD34⁺ hematopoietic stem cells, immature and mature Tcells and immature and mature B cells. The differentiation of T cellprecursors into a cytolytic T cell, when exposed to an antigen, issimilar to clonal selection of the immune system.

A “lymphoid cell” is a cell which is capable of producing an immuneresponse such as a cellular immune response, or a precursor cell of suchcell, and includes lymphocytes, preferably T lymphocytes, lymphoblasts,and plasma cells. A lymphoid cell may be an immune effector cell asdescribed herein. A preferred lymphoid cell is a T cell.

The terms “T cell” and “T lymphocyte” are used interchangeably hereinand include T helper cells (CD4+ T cells) and cytotoxic T cells (CTLs,CD8+ T cells) which comprise cytolytic T cells. The term“antigen-specific T cell” or similar terms relate to a T cell whichrecognizes the antigen to which the T cell is targeted and preferablyexerts effector functions of T cells.

T cells belong to a group of white blood cells known as lymphocytes, andplay a central role in cell-mediated immunity. They can be distinguishedfrom other lymphocyte types, such as B cells and natural killer cells bythe presence of a special receptor on their cell surface called T cellreceptor (TCR). The thymus is the principal organ responsible for thematuration of T cells. Several different subsets of T cells have beendiscovered, each with a distinct function.

T helper cells assist other white blood cells in immunologic processes,including maturation of B cells into plasma cells and activation ofcytotoxic T cells and macrophages, among other functions. These cellsare also known as CD4+ T cells because they express the CD4 glycoproteinon their surface. Helper T cells become activated when they arepresented with peptide antigens by MHC class II molecules that areexpressed on the surface of antigen presenting cells (APCs). Onceactivated, they divide rapidly and secrete small proteins calledcytokines that regulate or assist in the active immune response.

Cytotoxic T cells destroy virally infected cells and tumor cells, andare also implicated in transplant rejection. These cells are also knownas CD8+ T cells since they express the CD8 glycoprotein on theirsurface. These cells recognize their targets by binding to antigenassociated with MHC class I, which is present on the surface of nearlyevery cell of the body. A majority of T cells have a T cell receptor(TCR) existing as a complex of several proteins. The TCR of a T cell isable to interact with immunogenic peptides (epitopes) bound to majorhistocompatibility complex (MHC) molecules and presented on the surfaceof target cells. Specific binding of the TCR triggers a signal cascadeinside the T cell leading to proliferation and differentiation into amaturated effector T cell. The actual T cell receptor is composed of twoseparate peptide chains, which are produced from the independent T cellreceptor alpha and beta (TCRα and TCRβ) genes and are called α- andβ-TCR chains. γδ T cells (gamma delta T cells) represent a small subsetof T cells that possess a distinct T cell receptor (TCR) on theirsurface. However, in γδ T cells, the TCR is made up of one γ-chain andone δ-chain. This group of T cells is much less common (2% of total Tcells) than the αβ T cells.

“Humoral immunity” or “humoral immune response” is the aspect ofimmunity that is mediated by macromolecules found in extracellularfluids such as secreted antibodies, complement proteins, and certainantimicrobial peptides. It contrasts with cell-mediated immunity. Itsaspects involving antibodies are often called antibody-mediatedimmunity. Humoral immunity refers to antibody production and theaccessory processes that accompany it, including: Th2 activation andcytokine production, germinal center formation and isotype switching,affinity maturation and memory cell generation. It also refers to theeffector functions of antibodies, which include pathogen neutralization,classical complement activation, and opsonin promotion of phagocytosisand pathogen elimination.

In humoral immune response, first the B cells mature in the bone marrowand gain B-cell receptors (BCR's) which are displayed in large number onthe cell surface. These membrane-bound protein complexes have antibodieswhich are specific for antigen detection. Each B cell has a uniqueantibody that binds with an antigen. The mature B cells migrate from thebone marrow to the lymph nodes or other lymphatic organs, where theybegin to encounter pathogens. When a B cell encounters an antigen, theantigen is bound to the receptor and taken inside the B cell byendocytosis. The antigen is processed and presented on the B cell'ssurface again by MHC-II proteins. The B cell waits fora helper T cell(TH) to bind to the complex. This binding will activate the TH cell,which then releases cytokines that induce B cells to divide rapidly,making thousands of identical clones of the B cell. These daughter cellseither become plasma cells or memory cells. The memory B cells remaininactive here; later when these memory B cells encounter the sameantigen due to reinfection, they divide and form plasma cells. On theother hand, the plasma cells produce a large number of antibodies whichare released free into the circulatory system. These antibodies willencounter antigens and bind with them. This will either interfere withthe chemical interaction between host and foreign cells, or they mayform bridges between their antigenic sites hindering their properfunctioning, or their presence will attract macrophages or killer cellsto attack and phagocytose them.

The term “antibody” includes an immunoglobulin comprising at least twoheavy (H) chains and two light (L) chains inter-connected by disulfidebonds. Each heavy chain is comprised of a heavy chain variable region(abbreviated herein as VH) and a heavy chain constant region. Each lightchain is comprised of a light chain variable region (abbreviated hereinas VL) and a light chain constant region. The VH and VL regions can befurther subdivided into regions of hypervariability, termedcomplementarity determining regions (CDR), interspersed with regionsthat are more conserved, termed framework regions (FR). Each VH and VLis composed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. The variable regions of the heavy and light chains contain abinding domain that interacts with an antigen. The constant regions ofthe antibodies may mediate the binding of the immunoglobulin to hosttissues or factors, including various cells of the immune system (e.g.,effector cells) and the first component (Clq) of the classicalcomplement system. An antibody binds, preferably specifically binds withan antigen.

Antibodies expressed by B cells are sometimes referred to as the BCR (Bcell receptor) or antigen receptor. The five members included in thisclass of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the primaryantibody that is present in body secretions, such as saliva, tears,breast milk, gastrointestinal secretions and mucus secretions of therespiratory and genitourinary tracts. IgG is the most common circulatingantibody. IgM is the main immunoglobulin produced in the primary immuneresponse in most subjects. It is the most efficient immunoglobulin inagglutination, complement fixation, and other antibody responses, and isimportant in defense against bacteria and viruses. IgD is theimmunoglobulin that has no known antibody function, but may serve as anantigen receptor. IgE is the immunoglobulin that mediates immediatehypersensitivity by causing release of mediators from mast cells andbasophils upon exposure to allergen.

An “antibody heavy chain”, as used herein, refers to the larger of thetwo types of polypeptide chains present in antibody molecules in theirnaturally occurring conformations.

An “antibody light chain”, as used herein, refers to the smaller of thetwo types of polypeptide chains present in antibody molecules in theirnaturally occurring conformations, K and A light chains refer to the twomajor antibody light chain isotypes.

The present disclosure contemplates an immune response that may beprotective, preventive, prophylactic and/or therapeutic. As used herein,“induces [or inducing] an immune response” may indicate that no immuneresponse against a particular antigen was present before induction or itmay indicate that there was a basal level of immune response against aparticular antigen before induction, which was enhanced after induction.Therefore, “induces [or inducing] an immune response” includes “enhances[or enhancing] an immune response”. The term “immunotherapy” relates tothe treatment of a disease or condition by inducing, or enhancing animmune response. The term “immunotherapy” includes antigen immunizationor antigen vaccination.

The terms “immunization” or “vaccination” describe the process ofadministering an antigen to an individual with the purpose of inducingan immune response, for example, for therapeutic or prophylacticreasons.

The term “macrophage” refers to a subgroup of phagocytic cells producedby the differentiation of monocytes. Macrophages which are activated byinflammation, immune cytokines or microbial products nonspecificallyengulf and kill foreign pathogens within the macrophage by hydrolyticand oxidative attack resulting in degradation of the pathogen. Peptidesfrom degraded proteins are displayed on the macrophage cell surfacewhere they can be recognized by T cells, and they can directly interactwith antibodies on the B cell surface, resulting in T and B cellactivation and further stimulation of the immune response. Macrophagesbelong to the class of antigen presenting cells. In one embodiment, themacrophages are splenic macrophages.

The term “dendritic cell” (DC) refers to another subtype of phagocyticcells belonging to the class of antigen presenting cells. In oneembodiment, dendritic cells are derived from hematopoietic bone marrowprogenitor cells. These progenitor cells initially transform intoimmature dendritic cells. These immature cells are characterized by highphagocytic activity and low T cell activation potential. Immaturedendritic cells constantly sample the surrounding environment forpathogens such as viruses and bacteria. Once they have come into contactwith a presentable antigen, they become activated into mature dendriticcells and begin to migrate to the spleen or to the lymph node. Immaturedendritic cells phagocytose pathogens and degrade their proteins intosmall pieces and upon maturation present those fragments at their cellsurface using MHC molecules. Simultaneously, they upregulatecell-surface receptors that act as co-receptors in T cell activationsuch as CD80, CD86, and CD40 greatly enhancing their ability to activateT cells. They also upregulate CCR7, a chemotactic receptor that inducesthe dendritic cell to travel through the blood stream to the spleen orthrough the lymphatic system to a lymph node. Here they act asantigen-presenting cells and activate helper T cells and killer T cellsas well as B cells by presenting them antigens, alongside non-antigenspecific co-stimulatory signals. Thus, dendritic cells can activelyinduce a T cell- or B cell-related immune response. In one embodiment,the dendritic cells are splenic dendritic cells.

The term “antigen presenting cell” (APC) is a cell of a variety of cellscapable of displaying, acquiring, and/or presenting at least one antigenor antigenic fragment on (or at) its cell surface. Antigen-presentingcells can be distinguished in professional antigen presenting cells andnon-professional antigen presenting cells.

The term “professional antigen presenting cells” relates to antigenpresenting cells which constitutively express the MajorHistocompatibility Complex class II (MHC class II) molecules requiredfor interaction with naïve T cells. If a T cell interacts with the MHCclass II molecule complex on the membrane of the antigen presentingcell, the antigen presenting cell produces a co-stimulatory moleculeinducing activation of the T cell. Professional antigen presenting cellscomprise dendritic cells and macrophages.

The term “non-professional antigen presenting cells” relates to antigenpresenting cells which do not constitutively express MHC class IImolecules, but upon stimulation by certain cytokines such asinterferon-gamma. Exemplary, non-professional antigen presenting cellsinclude fibroblasts, thymic epithelial cells, thyroid epithelial cells,glial cells, pancreatic beta cells or vascular endothelial cells.

“Antigen processing” refers to the degradation of an antigen intoprocession products, which are fragments of said antigen (e.g., thedegradation of a protein into peptides) and the association of one ormore of these fragments (e.g., via binding) with MHC molecules forpresentation by cells, such as antigen presenting cells to specific Tcells.

The term “disease involving an antigen” refers to any disease whichimplicates an antigen, e.g. a disease which is characterized by thepresence of an antigen. The disease involving an antigen can be aninfectious disease. As mentioned above, the antigen may be adisease-associated antigen, such as a viral antigen. In one embodiment,a disease involving an antigen is a disease involving cells expressingan antigen, preferably on the cell surface.

The term “infectious disease” refers to any disease which can betransmitted from individual to individual or from organism to organism,and is caused by a microbial agent (e.g. common cold). Infectiousdiseases are known in the art and include, for example, a viral disease,a bacterial disease, or a parasitic disease, which diseases are causedby a virus, a bacterium, and a parasite, respectively. In this regard,the infectious disease can be, for example, hepatitis, sexuallytransmitted diseases (e.g. chlamydia or gonorrhea), tuberculosis,HIV/acquired immune deficiency syndrome (AIDS), diphtheria, hepatitis B,hepatitis C, cholera, severe acute respiratory syndrome (SARS), the birdflu, and influenza.

Exemplary Dosing Regimens

In some embodiments, compositions and methods disclosed herein can beused in accordance with an exemplary vaccination regimen as illustratedin FIG. 14 .

Primary Dosing Regimens

In some embodiments, subjects are administered a primary dosing regimen.A primary dosing regimen can comprise one or more doses. For example, insome embodiments, a primary dosing regimen comprises a single dose(PD₁). In some embodiments a primary dosing regimen comprises a firstdose (PD₁) and a second dose (PD₂). In some embodiments, a primarydosing regimen comprises a first dose, a second dose, and a third dose(PD₃). In some embodiments, a primary dosing regimen comprises a firstdose, a second dose, a third dose, and one or more additional doses(PD_(n)) of any one of the pharmaceutical compositions described herein.

In some embodiments, PD₁ comprises administering 1 to 100 ug of RNA. Insome embodiments, PD₁ comprises administering 1 to 60 ug of RNA In someembodiments, PD₁ comprises administering 1 to 50 ug of RNA. In someembodiments, PD₁ comprises administering 1 to 30 ug of RNA. In someembodiments, PD₁ comprises administering about 3 ug of RNA. In someembodiments, PD₁ comprises administering about 5 ug of RNA. In someembodiments, PD₁ comprises administering about 10 ug of RNA. In someembodiments, PD₁ comprises administering about 15 ug of RNA. In someembodiments, PD₁ comprises administering about 20 ug of RNA. In someembodiments, PD₁ comprises administering about ug of RNA. In someembodiments, PD₁ comprises administering about 50 ug of RNA. In someembodiments, PD₁ comprises administering about 60 ug of RNA.

In some embodiments, PD₂ comprises administering 1 to 100 ug of RNA. Insome embodiments, PD₂ comprises administering 1 to 60 ug of RNA. In someembodiments, PD₂ comprises administering 1 to 50 ug of RNA. In someembodiments, PD₂ comprises administering 1 to 30 ug of RNA. In someembodiments, PD₂ comprises administering about 3 ug. In someembodiments, PD₂ comprises administering about 5 ug of RNA. In someembodiments, PD₂ comprises administering about 10 ug of RNA. In someembodiments, PD₂ comprises administering about 15 ug of RNA. In someembodiments, PD₂ comprises administering about 20 ug RNA. In someembodiments, PD₂ comprises administering about ug of RNA. In someembodiments, PD₂ comprises administering about 50 ug of RNA. In someembodiments, PD₂ comprises administering about 60 ug of RNA.

In some embodiments, PD₃ comprises administering 1 to 100 ug of RNA. Insome embodiments, PD₃ comprises administering 1 to 60 ug of RNA. In someembodiments, PD₃ comprises administering 1 to 50 ug of RNA. In someembodiments, PD₃ comprises administering 1 to 30 ug of RNA. In someembodiments, PD₃ comprises administering about 3 ug of RNA. In someembodiments, PD₃ comprises administering about 5 ug of RNA. In someembodiments, PD₃ comprises administering about 10 ug of RNA. In someembodiments, PD₃ comprises administering about 15 ug of RNA. In someembodiments, PD₃ comprises administering about 20 ug of RNA. In someembodiments, PD₃ comprises administering about ug of RNA. In someembodiments, PD₃ comprises administering about 50 ug of RNA. In someembodiments, PD₃ comprises administering about 60 ug of RNA.

In some embodiments, PD_(n) comprises administering 1 to 100 ug of RNA.In some embodiments, PD_(n) comprises administering 1 to 60 ug of RNA.In some embodiments, PD_(n) comprises administering 1 to 50 ug of RNA.In some embodiments, PD_(n) comprises administering 1 to 30 ug of RNA.In some embodiments, PD_(n) comprises administering about 3 ug of RNA.In some embodiments, PD_(n) comprises administering about 5 ug of RNA.In some embodiments, PD_(n) comprises administering about 10 ug of RNA.In some embodiments, PD_(n) comprises administering about 15 ug of RNA.In some embodiments, PD_(n) comprises administering about 20 ug of RNA.In some embodiments, PD_(n) comprises administering about ug of RNA. Insome embodiments, PD_(n) comprises administering about 50 ug of RNA. Insome embodiments, PD_(n) comprises administering about 60 ug of RNA.

In some embodiments, PD₁ comprises an RNA encoding a SARS-CoV-2 Spikeprotein or an immunogenic fragment thereof from the Wuhan strain. Insome embodiments, PD₁ comprises an RNA encoding a Spike protein or animmunogenic fragment thereof from a SARS-CoV-2 strain that is prevalentand/or spreading rapidly in a relevant jurisdiction. In someembodiments, PD₁ comprises an RNA encoding a SARS-CoV-2 Spike protein oran immunogenic fragment thereof comprising one or more mutations from analpha variant. In some embodiments, PD₁ comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof comprisingone or more mutations from a delta variant. In some embodiments, PD₁comprises an RNA encoding a SARS-CoV-2 Spike protein or an immunogenicfragment thereof comprising one or more mutations from a beta variant.In some embodiments, PD₁ comprises an RNA encoding a SARS-CoV-2 Spikeprotein or an immunogenic fragment thereof comprising one or moremutations from an Omicron variant (e.g., a BA.4/5, BA.1, BA.2, XBB,XBB.1, or BQ.1 Omicron variant). In some embodiments, PD₁ comprises anRNA encoding a SARS-CoV-2 Spike protein or an immunogenic fragmentthereof from the Wuhan strain and one or more additional RNAs encoding aSpike protein or an immunogenic fragment thereof from a SARS-CoV-2strain that is prevalent and/or spreading rapidly in a relevantjurisdiction. In some embodiments, PD₁ comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from analpha variant. In some embodiments, PD₁ comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from abeta variant. In some embodiments, PD₁ comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from adelta variant. In some embodiments, PD₁ comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from anOmicron variant (e.g., a BA.4/5, BA.1, BA.2, XBB, XBB.1, or BQ.1 Omicronvariant).

In some embodiments, PD₂ comprises an RNA encoding a SARS-CoV-2 Spikeprotein or an immunogenic fragment thereof from the Wuhan strain. Insome embodiments, PD₂ comprises an RNA encoding a Spike protein or animmunogenic fragment thereof from a SARS-CoV-2 strain that is prevalentand/or spreading rapidly in a relevant jurisdiction. In someembodiments, PD₂ comprises an RNA encoding a SARS-CoV-2 Spike protein oran immunogenic fragment thereof comprising one or more mutations from analpha variant. In some embodiments, PD₂ comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof comprisingone or more mutations from a delta variant. In some embodiments, PD₂comprises an RNA encoding a SARS-CoV-2 Spike protein or an immunogenicfragment thereof comprising one or more mutations from a beta variant.In some embodiments, PD₂ comprises an RNA encoding a SARS-CoV-2 Spikeprotein or an immunogenic fragment thereof comprising one or moremutations from an Omicron variant (e.g., a BA.4/5, BA.1, BA.2, XBB,XBB.1, or BQ.1 Omicron variant). In some embodiments, PD₂ comprises anRNA encoding a SARS-CoV-2 Spike protein or an immunogenic fragmentthereof from the Wuhan strain and one or more additional RNAs encoding aSpike protein or an immunogenic fragment thereof from a SARS-CoV-2strain that is prevalent and/or spreading rapidly in a relevantjurisdiction. In some embodiments, PD₂ comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from analpha variant. In some embodiments, PD₂ comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from abeta variant. In some embodiments, PD₂ comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from adelta variant. In some embodiments, PD₂ comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from anOmicron variant (e.g., a BA.4/5, BA.1, BA.2, XBB, XBB.1, or BQ.1 Omicronvariant).

In some embodiments, PD₃ comprises an RNA encoding a SARS-CoV-2 Spikeprotein or an immunogenic fragment thereof from the Wuhan strain. Insome embodiments, PD₃ comprises an RNA encoding a Spike protein or animmunogenic fragment thereof from a SARS-CoV-2 strain that is prevalentand/or spreading rapidly in a relevant jurisdiction. In someembodiments, PD₃ comprises an RNA encoding a SARS-CoV-2 Spike protein oran immunogenic fragment thereof comprising one or more mutations from analpha variant. In some embodiments, PD₃ comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof comprisingone or more mutations from a delta variant. In some embodiments, PD₃comprises an RNA encoding a SARS-CoV-2 Spike protein or an immunogenicfragment thereof comprising one or more mutations from a beta variant.In some embodiments, PD₃ comprises an RNA encoding a SARS-CoV-2 Spikeprotein or an immunogenic fragment thereof comprising one or moremutations from an Omicron variant (e.g., a BA.4/5, BA.1, BA.2, XBB,XBB.1, or BQ.1 Omicron variant). In some embodiments, PD₃ comprises anRNA encoding a SARS-CoV-2 Spike protein or an immunogenic fragmentthereof from the Wuhan strain and one or more additional RNAs encoding aSpike protein or an immunogenic fragment thereof from a SARS-CoV-2strain that is prevalent and/or spreading rapidly in a relevantjurisdiction. In some embodiments, PD₃ comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from analpha variant. In some embodiments, PD₃ comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from abeta variant. In some embodiments, PD₃ comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from adelta variant. In some embodiments, PD₃ comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from anOmicron variant (e.g., a BA.4/5, BA.1, BA.2, XBB, XBB.1, or BQ.1 Omicronvariant).

In some embodiments, PD_(n) comprises an RNA encoding a SARS-CoV-2 Spikeprotein or an immunogenic fragment thereof from the Wuhan strain. Insome embodiments, PD_(n) comprises an RNA encoding a Spike protein or animmunogenic fragment thereof from a SARS-CoV-2 strain that is prevalentand/or spreading rapidly in a relevant jurisdiction. In someembodiments, PD_(n) comprises an RNA encoding a SARS-CoV-2 Spike proteinor an immunogenic fragment thereof comprising one or more mutations froman alpha variant. In some embodiments, PD_(n) comprises an RNA encodinga SARS-CoV-2 Spike protein or an immunogenic fragment thereof comprisingone or more mutations from a delta variant. In some embodiments, PD_(n)comprises an RNA encoding a SARS-CoV-2 Spike protein or an immunogenicfragment thereof comprising one or more mutations from a beta variant.In some embodiments, PD_(n) comprises an RNA encoding a SARS-CoV-2 Spikeprotein or an immunogenic fragment thereof comprising one or moremutations from an Omicron variant (e.g., a BA.4/5, BA.1, BA.2, XBB,XBB.1, or BQ.1 Omicron variant). In some embodiments, PD_(n) comprisesan RNA encoding a SARS-CoV-2 Spike protein or an immunogenic fragmentthereof from the Wuhan strain and one or more additional RNAs encoding aSpike protein or an immunogenic fragment thereof from a SARS-CoV-2strain that is prevalent and/or spreading rapidly in a relevantjurisdiction. In some embodiments, PD_(n) comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from analpha variant. In some embodiments, PD_(n) comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from abeta variant. In some embodiments, PD_(n) comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from adelta variant. In some embodiments, PD_(n) comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from anOmicron variant (e.g., a BA.4/5, BA.1, BA.2, XBB, XBB.1, or BQ.1 Omicronvariant).

In some embodiments, PD₁, PD₂, PD₃, and PD_(n) can each independentlycomprise a plurality of (e.g., at least two) mRNA compositions describedherein. In some embodiments PD₁, PD₂, PD₃, and PD_(n) can eachindependently comprise a first and a second mRNA composition. In someembodiments, at least one of a plurality of mRNA compositions comprisesBNT162b2 (e.g., as described herein). In some embodiments, at least oneof a plurality of mRNA compositions comprises an mRNA encoding aSARS-CoV-2 S protein or an immunogenic fragment thereof from a differentSARS-CoV-2 variant. In some embodiments, at least one of a plurality ofmRNA compositions comprises an mRNA encoding a SARS-CoV-2 S protein oran immunogenic fragment thereof from a Wuhan strain of SARS-CoV-2. Insome embodiments, at least one of a plurality of mRNA compositionscomprises an RNA encoding a SARS-CoV-2 S protein or an immunogenicfragment thereof comprising one or more mutations from a variant that isprevalent and/or spreading rapidly in a relevant jurisdiction. In someembodiments, at least one of a plurality of mRNA compositions comprisesan mRNA encoding a SARS-CoV-2 S protein or an immunogenic fragmentthereof comprising one or more mutations from an alpha variant. In someembodiments, at least one of a plurality of mRNA compositions comprisesan mRNA encoding a SARS-CoV-2 S protein or an immunogenic fragmentthereof comprising one or more mutations from a delta variant. In someembodiments, at least one of a plurality of mRNA compositions comprisesan mRNA encoding a SARS-CoV-2 S protein or an immunogenic fragmentthereof comprising one or more mutations from an Omicron variant (e.g.,a BA.4/5, BA.1, BA.2, XBB, XBB.1, or BQ.1 Omicron variant).

In some embodiments, a plurality of mRNA compositions given in PD₁, PD₂,PD₃, and/or PD_(n) can each independently comprise at least twodifferent mRNA constructs (e.g., differing in at protein-encodingsequences). For example, in some embodiments a plurality of mRNAcompositions given in PD₁, PD₂, PD₃, and/or PD_(n) can eachindependently comprise an mRNA encoding a SARS-CoV-2 S protein or animmunogenic fragment thereof from a Wuhan strain of SARS-CoV-2 and anmRNA encoding a SARS-CoV-2 S protein or an immunogenic fragment thereofcomprising one or more mutations from a variant that is prevalent and/orspreading rapidly in a relevant jurisdiction. In some embodiments aplurality of mRNA compositions given in PD₁, PD₂, PD₃, and/or PD_(n) caneach independently comprise an mRNA encoding a SARS-CoV-2 S protein oran immunogenic fragment thereof derived from a Wuhan strain ofSARS-CoV-2 and an mRNA encoding a SARS-CoV-2 S protein or an immunogenicfragment thereof comprising one or more mutations from a variant that isprevalent and/or spreading rapidly in a relevant jurisdiction. In somesuch embodiments, a variant can be an alpha variant. In some suchembodiments, a variant can be a delta variant. In some such embodimentsa variant can be an Omicron variant (e.g., a BA.4/5, BA.1, BA.2, XBB,XBB.1, or BQ.1 Omicron variant). In some embodiments, each of aplurality of mRNA compositions given in PD₁, PD₂, PD₃, and/or PD_(n) canindependently comprise at least two mRNAs, each encoding a SARS-CoV-2 Sprotein or an immunogenic fragment thereof comprising one or moremutations from a distinct variant that is prevalent and/or spreadingrapidly in a relevant jurisdiction. In some embodiments, each of aplurality of mRNA compositions given in PD₁, PD₂, PD₃, and/or PD_(n) canindependently comprise an mRNA encoding a SARS-CoV-2 S protein or animmunogenic fragment thereof from an alpha variant and an mRNA encodinga SARS-CoV-2 S protein or an immunogenic fragment thereof comprising oneor more mutations from a delta variant. In some embodiments, each of aplurality of mRNA compositions given in PD₁, PD₂, PD₃, and/or PD_(n) canindependently comprise an mRNA encoding a SARS-CoV-2 S protein or animmunogenic fragment thereof from an alpha variant and an mRNA encodinga SARS-CoV-2 S protein or an immunogenic fragment thereof comprising oneor more mutations from an Omicron variant (e.g., a BA.4/5, BA.1, BA.2,XBB, XBB.1, or BQ.1 Omicron variant). In some embodiments, each of aplurality of mRNA compositions given in PD₁, PD₂, PD₃, and/or PD_(n) canindependently comprise an mRNA encoding a SARS-CoV-2 S protein or animmunogenic fragment thereof from a delta variant and an mRNA encoding aSARS-CoV-2 S protein or an immunogenic fragment thereof comprising oneor more mutations from an Omicron variant (e.g., a BA.4/5, BA.1, BA.2,XBB, XBB.1, or BQ.1 Omicron variant).

In some embodiments, PD₁, PD₂, PD₃, and/or PD_(n) each comprise aplurality of mRNA compositions, wherein each mRNA composition isseparately administered to a subject. For example, in some embodimentseach mRNA composition is administered via intramuscular injection atdifferent injection sites. For example, in some embodiments, a first andsecond mRNA composition given in PD₁, PD₂, PD₃, and/or PD_(n) areseparately administered to different arms of a subject via intramuscularinjection.

In some embodiments, PD₁, PD₂, PD₃, and/or PD_(n) comprise administeringa plurality of RNA molecules, wherein each RNA molecule encodes a Spikeprotein comprising mutations from a different SARS-CoV-2 variant, andwherein the plurality of RNA molecules are administered to the subjectin a single formulation. In some embodiments, the single formulationcomprises an RNA encoding a Spike protein or an immunogenic variantthereof from the Wuhan strain and an RNA encoding a SARS-CoV-2 Spikeprotein or an immunogenic fragment thereof comprising one or moremutations from an alpha variant. In some embodiments, the singleformulation comprises an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof from the Wuhan strain and an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof comprisingone or more mutations from a beta variant. In some embodiments, thesingle formulation comprises an RNA encoding a SARS-CoV-2 Spike proteinor an immunogenic fragment thereof from the Wuhan strain and an RNAencoding a SARS-CoV-2 Spike protein or an immunogenic fragment thereofcomprising one or more mutations from a delta variant. In someembodiments, the single formulation comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from anOmicron variant (e.g., a BA.4/5, BA.1, BA.2, XBB, XBB.1, or BQ.1 Omicronvariant).

In some embodiments, the length of time between PD₁ and PD₂ (PI₁) is atleast about 1 week, at least about 2 weeks, at least about 3 weeks, orat least about 4 weeks. In some embodiments, PI₁ is about 1 week toabout 12 weeks. In some embodiments, PI₁ is about 1 week to about 10weeks. In some embodiments, PI₁ is about 2 weeks to about 10 weeks. Insome embodiments, PI₁ is about 2 weeks to about 8 weeks. In someembodiments, PI₁ is about 3 weeks to about 8 weeks. In some embodiments,PI₁ is about 4 weeks to about 8 weeks. In some embodiments, PI₁ is about6 weeks to about 8 weeks. In some embodiments PI₁ is about 3 to about 4weeks. In some embodiments, PI₁ is about 1 week. In some embodiments,PI₁ is about 2 weeks. In some embodiments, PI₁ is about 3 weeks. In someembodiments, PI₁ is about 4 weeks. In some embodiments, PI₁ is about 5weeks. In some embodiments, PI₁ is about 6 weeks. In some embodiments,PI₁ is about 7 weeks. In some embodiments, PI₁ is about 8 weeks. In someembodiments, PI₁ is about 9 weeks. In some embodiments, PI₁ is about 10weeks. In some embodiments, PI₁ is about 11 weeks. In some embodiments,PI₁ is about 12 weeks.

In some embodiments, the length of time between PD₂ and PD₃ (PI₂) is atleast about 1 week, at least about 2 weeks, or at least about 3 weeks.In some embodiments, PI₂ is about 1 week to about 12 weeks. In someembodiments, PI₂ is about 1 week to about 10 weeks. In some embodiments,PI₂ is about 2 weeks to about 10 weeks. In some embodiments, PI₂ isabout 2 weeks to about 8 weeks. In some embodiments, PI₂ is about 3weeks to about 8 weeks. In some embodiments, PI₂ is about 4 weeks toabout 8 weeks. In some embodiments, PI₂ is about 6 weeks to about 8weeks. In some embodiments PI₂ is about 3 to about 4 weeks. In someembodiments, PI₂ is about 1 week. In some embodiments, PI₂ is about 2weeks. In some embodiments, PI₂ is about 3 weeks. In some embodiments,PI₂ is about 4 weeks. In some embodiments, PI₂ is about 5 weeks. In someembodiments, PI₂ is about 6 weeks. In some embodiments, PI₂ is about 7weeks. In some embodiments, PI₂ is about 8 weeks. In some embodiments,PI₂ is about 9 weeks. In some embodiments, PI₂ is about 10 weeks. Insome embodiments, PI₂ is about 11 weeks. In some embodiments, PI₂ isabout 12 weeks.

In some embodiments, the length of time between PD₃ and a subsequentdose that is part of the Primary Dosing Regimen, or between doses forany dose beyond PD₃ (PI_(n)) is each separately and independentlyselected from: about 1 week or more, about 2 weeks or more, or about 3weeks or more. In some embodiments, PI_(n) is about 1 week to about 12weeks. In some embodiments, PI_(n) is about 1 week to about 10 weeks. Insome embodiments, PI_(n) is about 2 weeks to about 10 weeks. In someembodiments, PI_(n) is about 2 weeks to about 8 weeks. In someembodiments, PI_(n) is about 3 weeks to about 8 weeks. In someembodiments, PI_(n) is about 4 weeks to about 8 weeks. In someembodiments, PI_(n) is about 6 weeks to about 8 weeks. In someembodiments PI_(n) is about 3 to about 4 weeks. In some embodiments, PI₂is about 1 week. In some embodiments, PI_(n) is about 2 weeks. In someembodiments, PI_(n) is about 3 weeks. In some embodiments, PI_(n) isabout 4 weeks. In some embodiments, PI_(n) is about 5 weeks. In someembodiments, PI_(n) is about 6 weeks. In some embodiments, PI_(n) isabout 7 weeks. In some embodiments, PI_(n) is about 8 weeks. In someembodiments, PI_(n) is about 9 weeks. In some embodiments, PI_(n) isabout 10 weeks. In some embodiments, PI_(n) is about 11 weeks. In someembodiments, PI_(n) is about 12 weeks.

In some embodiments, one or more compositions adminstered in PD₁ areformulated in a Tris buffer. In some embodiments, one or morecompositions administered in PD₂ are formulated in a Tris buffer. Insome embodiments, one or more compositions administering in PD₃ areformulated in a Tris buffer. In some embodiments, one or morecompositions administered in PD_(n) are formulated in a Tris buffer.

In some embodiments, the primary dosing regimen comprises administeringtwo or more mRNA compositions described herein, and at least two of themRNA compositions have different formulations. In some embodiments, theprimary dosing regimen comprises PD₁ and PD₂, where PD₁ comprisesadministering an mRNA formulated in a Tris buffer and PD₂ comprisesadministering an mRNA formulated in a PBS buffer. In some embodiments,the primary dosing regimen comprises PD₁ and PD₂, where PD₁ comprisesadministering an mRNA formulated in a PBS buffer and PD₂ comprisesadministering an mRNA formulated in a Tris buffer.

In some embodiments, one or more mRNA compositions given in PD₁, PD₂,PD₃, and/or PD_(n) can be administered in combination with anothervaccine. In some embodiments, another vaccine is for a disease that isnot COVID-19. In some embodiments, the disease is one that increasesdeleterious effects of SARS-CoV-2 when a subject is coinfected with thedisease and SARS-CoV-2. In some embodiments, the disease is one thatincreases the transmission rate of SARS-CoV-2 when a subject iscoinfected with the disease and SARS-CoV-2. In some embodiments, anothervaccine is a different commerically available vaccine. In someembodiments, the different commercially available vaccine is an RNAvaccine. In some embodiments, the different commercially availablevaccine is a polypeptide-based vaccine. In some embodiments, anothervaccine (e.g., as described herein) and one or more mRNA compositionsgiven in PD₁, PD₂, PD₃, and/or PD_(n) are separately administered, forexample, in some embodiments via intramuscular injection, at differentinjection sites. For example, in some embodiments, an influenza vaccineand one or more SARS-CoV-2 mRNA compositions described herein given inPD₁, PD₂, PD₃, and/or PD_(n) are separately administered to differentarms of a subject via intramuscular injection.

Booster Dosing Regimens

In some embodiments, methods of vaccination disclosed herein compriseone or more Booster Dosing Regimens. The Booster Dosing Regimensdisclosed herein comprise one or more doses. In some embodiments, aBooster Dosing Regimen is administered to patients who have beenadministered a Primary Dosing Regimen (e.g., as described herein). Insome embodiments a Booster Dosing Regimen is administered to patientswho have not received a pharmaceutical composition disclosed herein. Insome embodiments a Booster Dosing Regimen is administered to patientswho have been previously vaccinated with a COVID-19 vaccine that isdifferent from the vaccine administered in a Primary Dosing Regimen.

In some embodiments, the length of time between the Primary DosingRegimen and the Booster Dosing Regimen is at least 1 week, at least 2weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at leastweeks, at least 11 weeks, at least 12 weeks, at least 2 months, at least3 months, at least 4 months, at least 5 months, at least 6 months, atleast 7 months, at least 8 months, at least 9 months, at least 10months, at least 11 months, or at least 12 months or longer. In someembodiments, the length of time between the Primary Dosing Regimen andthe Booster Dosing Regimen is about 1 month. In some embodiments, thelength of time between the Primary Dosing Regimen and the Booster DosingRegimen is at least about 2 months. In some embodiments, the length oftime between the Primary Dosing Regimen and the Booster Dosing Regimenis at least about 3 months. In some embodiments, the length of timebetween the Primary Dosing Regimen and the Booster Dosing Regimen is atleast about 4 months. In some embodiments, the length of time betweenthe Primary Dosing Regimen and the Booster Dosing Regimen is at leastabout 5 months. In some embodiments, the length of time between thePrimary Dosing Regimen and the Booster Dosing Regimen is at least about6 months. In some embodiments, the length of time between the PrimaryDosing Regimen and the Booster Dosing Regimen is from about 1 month toabout 48 months. In some embodiments, the length of time between thePrimary Dosing Regimen and the Booster Dosing Regimen is from about 1month to about 36 months. In some embodiments, the length of timebetween the primary dosing regimen and the Booster Dosing Regimen isfrom about 1 month to about 24 months. In some embodiments, the lengthof time between the Primary Dosing Regimen and the Booster DosingRegimen is from about 2 months to about 24 months. In some embodiments,the length of time between the Primary Dosing Regimen and the BoosterDosing Regimen is from about 3 months to about 24 months. In someembodiments, the length of time between the primary dosing regimen andthe Booster Dosing Regimen is from about 3 months to about 18 months. Insome embodiments, the length of time between the primary dosing regimenand the Booster Dosing Regimen is from about 3 months to about 12months. In some embodiments, the length of time between the primarydosing regimen and the Booster Dosing Regimen is from about 6 months toabout 12 months. In some embodiments, the length of time between thePrimary Dosing Regimen and the Booster Dosing Regimen is from about 3months to about 9 months. In some embodiments, the length of timebetween the Primary Dosing Regimen and the Booster Dosing Regimen isfrom about 5 months to about 7 months. In some embodiments, the lengthof time between the Primary Dosing Regimen and the Booster DosingRegimen is about 6 months.

In some embodiments, subjects are administered a Booster Dosing Regimen.A Booster dosing regimen can comprise one or more doses. For example, insome embodiments, a Booster Dosing Regimen comprises a single dose(BD₁). In some embodiments a Booster Dosing Regimen comprises a firstdose (BD₁) and a second dose (BD₂). In some embodiments, a BoosterDosing Regimen comprises a first dose, a second dose, and a third dose(BD₃). In some embodiments, a Booster Dosing Regimen comprises a firstdose, a second dose, a third dose, and one or more additional doses(BD_(n)) of any one of the pharmaceutical compositions described herein.

In some embodiments, BD₁ comprises administering 1 to 100 ug of RNA. Insome embodiments, BD₁ comprises administering 1 to 60 ug of RNA. In someembodiments, BD₁ comprises administering 1 to 50 ug of RNA. In someembodiments, BD₁ comprises administering 1 to 30 ug of RNA. In someembodiments, BD₁ comprises administering about 3 ug of RNA. In someembodiments, BD₁ comprises administering about 5 ug of RNA. In someembodiments, BD₁ comprises administering about 10 ug of RNA. In someembodiments, BD₁ comprises administering about 15 ug of RNA. In someembodiments, BD₁ comprises administering about 20 ug of RNA. In someembodiments, BD₁ comprises administering about ug of RNA. In someembodiments, BD₁ comprises administering about 50 ug of RNA. In someembodiments, BD₁ comprises administering about 60 ug of RNA.

In some embodiments, BD₂ comprises administering 1 to 100 ug of RNA. Insome embodiments, BD₂ comprises administering 1 to 60 ug of RNA. In someembodiments, BD₂ comprises administering 1 to 50 ug of RNA. In someembodiments, BD₂ comprises administering 1 to 30 ug of RNA. In someembodiments, BD₂ comprises administering about 3 ug. In someembodiments, BD₂ comprises administering about 5 ug of RNA. In someembodiments, BD₂ comprises administering about 10 ug of RNA. In someembodiments, BD₂ comprises administering about 15 ug of RNA. In someembodiments, BD₂ comprises administering about 20 ug RNA. In someembodiments, BD₂ comprises administering about ug of RNA. In someembodiments, BD₂ comprises administering about 50 ug of RNA. In someembodiments, BD₂ comprises administering about 60 ug of RNA.

In some embodiments, BD₃ comprises administering 1 to 100 ug of RNA. Insome embodiments, BD₃ comprises administering 1 to 60 ug of RNA. In someembodiments, BD₃ comprises administering 1 to 50 ug of RNA. In someembodiments, BD₃ comprises administering 1 to 30 ug of RNA. In someembodiments, BD₃ comprises administering about 3 ug of RNA. In someembodiments, BD₃ comprises administering about 5 ug of RNA. In someembodiments, BD₃ comprises administering about 10 ug of RNA. In someembodiments, BD₃ comprises administering about 15 ug of RNA. In someembodiments, BD₃ comprises administering about 20 ug of RNA. In someembodiments, BD₃ comprises administering about ug of RNA. In someembodiments, BD₃ comprises administering about 50 ug of RNA. In someembodiments, BD₃ comprises administering about 60 ug of RNA.

In some embodiments, BD_(n) comprises administering 1 to 100 ug of RNA.In some embodiments, BD_(n) comprises administering 1 to 60 ug of RNA.In some embodiments, BD_(n) comprises administering 1 to 50 ug of RNA.In some embodiments, BD_(n) comprises administering 1 to 30 ug of RNA.In some embodiments, BD_(n) comprises administering about 3 ug of RNA.In some embodiments, BD_(n) comprises administering about 5 ug of RNA.In some embodiments, BD_(n) comprises administering about 10 ug of RNA.In some embodiments, BD_(n) comprises administering about 15 ug of RNA.In some embodiments, BD_(n) comprises administering about 20 ug of RNA.In some embodiments, BD_(n) comprises administering about ug of RNA. Insome embodiments, BD_(n) comprises administering about 60 ug of RNA. Insome embodiments, BD_(n) comprises administering about 50 ug of RNA.

In some embodiments, BD₁ comprises an RNA encoding a SARS-CoV-2 Spikeprotein or an immunogenic fragment thereof from the Wuhan strain. Insome embodiments, BD₁ comprises an RNA encoding a Spike protein or animmunogenic fragment thereof from a SARS-CoV-2 strain that is prevalentand/or spreading rapidly in a relevant jurisdiction. In someembodiments, BD₁ comprises an RNA encoding a SARS-CoV-2 Spike protein oran immunogenic fragment thereof comprising one or more mutations from analpha variant. In some embodiments, BD₁ comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof comprisingone or more mutations from a delta variant. In some embodiments, BD₁comprises an RNA encoding a SARS-CoV-2 Spike protein or an immunogenicfragment thereof comprising one or more mutations from a beta variant.In some embodiments, BD₁ comprises an RNA encoding a SARS-CoV-2 Spikeprotein or an immunogenic fragment thereof comprising one or moremutations from an Omicron variant (e.g., a BA.4/5, BA.1, BA.2, XBB,XBB.1, or BQ.1 Omicron variant).

In some embodiments, BD₁ comprises an RNA encoding a SARS-CoV-2 Spikeprotein or an immunogenic fragment thereof from the Wuhan strain and oneor more RNA encoding a Spike protein or an immunogenic fragment thereoffrom a SARS-CoV-2 strain that is prevalent and/or spreading rapidly in arelevant jurisdiction. In some embodiments, BD₁ comprises an RNAencoding a SARS-CoV-2 Spike protein or an immunogenic fragment thereoffrom the Wuhan strain and an RNA encoding a SARS-CoV-2 Spike protein oran immunogenic fragment thereof comprising one or more mutations from aalpha variant. In some embodiments, BD, comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from adelta variant. In some embodiments, BD₁ comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from abeta variant. In some embodiments, BD₁ comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from anOmicron variant (e.g., a BA.4/5, BA.1, BA.2, XBB, XBB.1, or BQ.1 Omicronvariant).

In some embodiments, BD₂ comprises an RNA encoding a SARS-CoV-2 Spikeprotein or an immunogenic fragment thereof from the Wuhan strain. Insome embodiments, BD₂ comprises an RNA encoding a Spike protein or animmunogenic fragment thereof from a SARS-CoV-2 strain that is prevalentand/or spreading rapidly in a relevant jurisdiction. In someembodiments, BD₂ comprises an RNA encoding a SARS-CoV-2 Spike protein oran immunogenic fragment thereof comprising one or more mutations from analpha variant. In some embodiments, BD₂ comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof comprisingone or more mutations from a delta variant. In some embodiments, BD₂comprises an RNA encoding a SARS-CoV-2 Spike protein or an immunogenicfragment thereof comprising one or more mutations from a beta variant.In some embodiments, BD₂ comprises an RNA encoding a SARS-CoV-2 Spikeprotein or an immunogenic fragment thereof comprising one or moremutations from an Omicron variant (e.g., a BA.4/5, BA.1, BA.2, XBB,XBB.1, or BQ.1 Omicron variant). In some embodiments, BD₂ comprises anRNA encoding a SARS-CoV-2 Spike protein or an immunogenic fragmentthereof from the Wuhan strain and one or more RNA encoding a Spikeprotein or an immunogenic fragment thereof from a SARS-CoV-2 strain thatis prevalent and/or spreading rapidly in a relevant jurisdiction. Insome embodiments, BD₂ comprises an RNA encoding a SARS-CoV-2 Spikeprotein or an immunogenic fragment thereof from the Wuhan strain and anRNA encoding a SARS-CoV-2 Spike protein or an immunogenic fragmentthereof comprising one or more mutations from a alpha variant. In someembodiments, BD₂ comprises an RNA encoding a SARS-CoV-2 Spike protein oran immunogenic fragment thereof from the Wuhan strain and an RNAencoding a SARS-CoV-2 Spike protein or an immunogenic fragment thereofcomprising one or more mutations from a delta variant. In someembodiments, BD₂ comprises an RNA encoding a SARS-CoV-2 Spike protein oran immunogenic fragment thereof from the Wuhan strain and an RNAencoding a SARS-CoV-2 Spike protein or an immunogenic fragment thereofcomprising one or more mutations from a beta variant. In someembodiments, BD₂ comprises an RNA encoding a SARS-CoV-2 Spike protein oran immunogenic fragment thereof from the Wuhan strain and an RNAencoding a SARS-CoV-2 Spike protein or an immunogenic fragment thereofcomprising one or more mutations from an Omicron variant (e.g., aBA.4/5, BA.1, BA.2, XBB, XBB.1, or BQ.1 Omicron variant).

In some embodiments, BD₃ comprises an RNA encoding a SARS-CoV-2 Spikeprotein or an immunogenic fragment thereof from the Wuhan strain. Insome embodiments, BD₃ comprises an RNA encoding a Spike protein or animmunogenic fragment thereof from a SARS-CoV-2 strain that is prevalentand/or spreading rapidly in a relevant jurisdiction. In someembodiments, BD₃ comprises an RNA encoding a SARS-CoV-2 Spike protein oran immunogenic fragment thereof comprising one or more mutations from analpha variant. In some embodiments, BD₃ comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof comprisingone or more mutations from a delta variant. In some embodiments, BD₃comprises an RNA encoding a SARS-CoV-2 Spike protein or an immunogenicfragment thereof comprising one or more mutations from a beta variant.In some embodiments, BD₃ comprises an RNA encoding a SARS-CoV-2 Spikeprotein or an immunogenic fragment thereof comprising one or moremutations from an Omicron variant (e.g., a BA.4/5, BA.1, BA.2, XBB,XBB.1, or BQ.1 Omicron variant).

In some embodiments, BD₃ comprises an RNA encoding a SARS-CoV-2 Spikeprotein or an immunogenic fragment thereof from the Wuhan strain and oneor more RNA encoding a Spike protein or an immunogenic fragment thereoffrom a SARS-CoV-2 strain that is prevalent and/or spreading rapidly in arelevant jurisdiction. In some embodiments, BD₃ comprises an RNAencoding a SARS-CoV-2 Spike protein or an immunogenic fragment thereoffrom the Wuhan strain and an RNA encoding a SARS-CoV-2 Spike protein oran immunogenic fragment thereof comprising one or more mutations from aalpha variant. In some embodiments, BD₃ comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from adelta variant. In some embodiments, BD₃ comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from abeta variant. In some embodiments, BD₃ comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from anOmicron variant (e.g., a BA.4/5, BA.1, BA.2, XBB, XBB.1, or BQ.1 Omicronvariant).

In some embodiments, BD_(n) comprises an RNA encoding a SARS-CoV-2 Spikeprotein or an immunogenic fragment thereof from the Wuhan strain. Insome embodiments, BD_(n) comprises an RNA encoding a Spike protein or animmunogenic fragment thereof from a SARS-CoV-2 strain that is prevalentand/or spreading rapidly in a relevant jurisdiction. In someembodiments, BD_(n) comprises an RNA encoding a SARS-CoV-2 Spike proteinor an immunogenic fragment thereof comprising one or more mutations froman alpha variant. In some embodiments, BD_(n) comprises an RNA encodinga SARS-CoV-2 Spike protein or an immunogenic fragment thereof comprisingone or more mutations from a delta variant. In some embodiments, BD_(n)comprises an RNA encoding a SARS-CoV-2 Spike protein or an immunogenicfragment thereof comprising one or more mutations from a beta variant.In some embodiments, BD_(n) comprises an RNA encoding a SARS-CoV-2 Spikeprotein or an immunogenic fragment thereof comprising one or moremutations from an Omicron variant (e.g., a BA.4/5, BA.1, BA.2, XBB,XBB.1, or BQ.1 Omicron variant).

In some embodiments, BD_(n) comprises an RNA encoding a SARS-CoV-2 Spikeprotein or an immunogenic fragment thereof from the Wuhan strain and oneor more RNA encoding a Spike protein or an immunogenic fragment thereoffrom a SARS-CoV-2 strain that is prevalent and/or spreading rapidly in arelevant jurisdiction. In some embodiments, BD_(n) comprises an RNAencoding a SARS-CoV-2 Spike protein or an immunogenic fragment thereoffrom the Wuhan strain and an RNA encoding a SARS-CoV-2 Spike protein oran immunogenic fragment thereof comprising one or more mutations from aalpha variant. In some embodiments, BD_(n) comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from adelta variant. In some embodiments, BD_(n) comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from abeta variant. In some embodiments, BD_(n) comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from anOmicron variant (e.g., a BA.4/5, BA.1, BA.2, XBB, XBB.1, or BQ.1 Omicronvariant).

In some embodiments, BD₁, BD₂, BD₃, and BD_(n) can each independentlycomprise a plurality of (e.g., at least two) mRNA compositions describedherein. In some embodiments BD₁, BD₂, BD₃, and BD_(n) can eachindependently comprise a first and a second mRNA composition. In someembodiments, BD₁, BD₂, BD₃, and BD_(n) can each independently comprise aplurality of (e.g., at least two) mRNA compositions, wherein, at leastone of the plurality of mRNA compositions comprises BNT162b2 (e.g., asdescribed herein). In some embodiments, at least one of a plurality ofmRNA compositions comprises an mRNA encoding a SARS-CoV-2 S protein oran immunogenic fragment thereof from a different SARS-CoV-2 variant(e.g., a variant that is prevalent or rapidly spreading in a relevantjurisdiction, e.g., a variant disclosed herein). In some embodiments, atleast one of a plurality of mRNA compositions comprises an mRNA encodinga SARS-CoV-2 S protein or an immunogenic fragment thereof from a Wuhanstrain of SARS-CoV-2. In some embodiments, at least one of a pluralityof mRNA compositions comprises an RNA encoding a SARS-CoV-2 S protein oran immunogenic fragment thereof comprising one or more mutations from avariant that is prevalent and/or spreading rapidly in a relevantjurisdiction. In some embodiments, at least one of a plurality of mRNAcompositions comprises an mRNA encoding a SARS-CoV-2 S protein or animmunogenic fragment thereof comprising one or more mutations from analpha variant. In some embodiments, at least one of a plurality of mRNAcompositions comprises an mRNA encoding a SARS-CoV-2 S protein or animmunogenic fragment thereof comprising one or more mutations from adelta variant. In some embodiments, at least one of a plurality of mRNAcompositions comprises an mRNA encoding a SARS-CoV-2 S protein or animmunogenic fragment thereof comprising one or more mutations from anOmicron variant (e.g., a BA.4/5, BA.1, BA.2, XBB, XBB.1, or BQ.1 Omicronvariant).

In some embodiments, a plurality of mRNA compositions given in BD₁, BD₂,BD₃, and/or BD_(n) can each independently comprise at least twodifferent mRNA constructs (e.g., mRNA constructs having differingprotein-encoding sequences). For example, in some embodiments aplurality of mRNA compositions given in BD₁, BD₂, BD₃, and/or BD_(n) caneach independently comprise an mRNA encoding a SARS-CoV-2 S protein oran immunogenic fragment thereof from a Wuhan strain of SARS-CoV-2 and anmRNA encoding a SARS-CoV-2 S protein or an immunogenic fragment thereofcomprising one or more mutations from a variant that is prevalent and/orspreading rapidly in a relevant jurisdiction. In some embodiments aplurality of mRNA compositions given in BD₁, BD₂, BD₃, and/or BD_(n) caneach independently comprise an mRNA encoding a SARS-CoV-2 S protein oran immunogenic fragment thereof derived from a Wuhan strain ofSARS-CoV-2 and an mRNA encoding a SARS-CoV-2 S protein or an immunogenicfragment thereof comprising one or more mutations from a variant that isprevalent and/or spreading rapidly in a relevant jurisdiction. In somesuch embodiments, a variant can be an alpha variant. In some suchembodiments, a variant can be a delta variant. In some such embodimentsa variant can be an Omicron variant (e.g., a BA.4/5, BA.1, BA.2, XBB,XBB.1, or BQ.1 Omicron variant).

In some embodiments, a plurality of mRNA compositions given in BD₁, BD₂,BD₃, and/or BD_(n) can each independently comprise at least two mRNAseach encoding a SARS-CoV-2 S protein or an immunogenic fragment thereofcomprising one or more mutations from a distinct variant that isprevalent and/or spreading rapidly in a relevant jurisdiction. In someembodiments a plurality of mRNA compositions given in BD₁, BD₂, BD₃,and/or BD_(n) can each independently comprise an mRNA encoding aSARS-CoV-2 S protein or an immunogenic fragment thereof from an alphavariant and an mRNA encoding a SARS-CoV-2 S protein or an immunogenicfragment thereof comprising one or more mutations from a delta variant.In some embodiments a plurality of mRNA compositions given in BD₁, BD₂,BD₃, and/or BD_(n) can each independently comprise an mRNA encoding aSARS-CoV-2 S protein or an immunogenic fragment thereof from an alphavariant and an mRNA encoding a SARS-CoV-2 S protein or an immunogenicfragment thereof comprising one or more mutations from an Omicronvariant (e.g., a BA.4/5, BA.1, BA.2, XBB, XBB.1, or BQ.1 Omicronvariant). In some embodiments a plurality of mRNA compositions given inBD₁, BD₂, BD₃, and/or BD_(n) can each independently comprise an mRNAencoding a SARS-CoV-2 S protein or an immunogenic fragment thereof froma delta variant and an mRNA encoding a SARS-CoV-2 S protein or animmunogenic fragment thereof comprising one or more mutations from anOmicron variant (e.g., a BA.4/5, BA.1, BA.2, XBB, XBB.1, or BQ.1 Omicronvariant).

In some embodiments, a plurality of mRNA compositions given in BD₁, BD₂,BD₃, and/or BD₁ are separately administered to a subject, for example,in some embodiments via intramuscular injection, at different injectionsites. For example, in some embodiments, a first and second mRNAcomposition given in BD₁, BD₂, BD₃, and/or BD_(n) are separatelyadministered to different arms of a subject via intramuscular injection.

In some embodiments, the length of time between BD₁ and BD₂ (BI₁) is atleast about 1 week, at least about 2 weeks, at least about 3 weeks, orat least about 4 weeks. In some embodiments, BI₁ is about 1 week toabout 12 weeks. In some embodiments, BI₁ is about 1 week to about 10weeks. In some embodiments, BI₁ is about 2 weeks to about 10 weeks. Insome embodiments, BI₁ is about 2 weeks to about 8 weeks. In someembodiments, BI₁ is about 3 weeks to about 8 weeks. In some embodiments,BI₁ is about 4 weeks to about 8 weeks. In some embodiments, BI₁ is about6 weeks to about 8 weeks. In some embodiments BI₁ is about 3 to about 4weeks. In some embodiments, BI₁ is about 1 week. In some embodiments,BI₁ is about 2 weeks. In some embodiments, BI₁ is about 3 weeks. In someembodiments, BI₁ is about 4 weeks. In some embodiments, BI₁ is about 5weeks. In some embodiments, BI₁ is about 6 weeks. In some embodiments,BI₁ is about 7 weeks. In some embodiments, BI₁ is about 8 weeks. In someembodiments, BI₁ is about 9 weeks. In some embodiments, BI₁ is about 10weeks.

In some embodiments, the length of time between BD₂ and BD₃ (BI₂) is atleast about 1 week, at least about 2 weeks, or at least about 3 weeks.In some embodiments, BI₂ is about 1 week to about 12 weeks. In someembodiments, BI₂ is about 1 week to about 10 weeks. In some embodiments,BI₂ is about 2 weeks to about 10 weeks. In some embodiments, BI₂ isabout 2 weeks to about 8 weeks. In some embodiments, BI₂ is about 3weeks to about 8 weeks. In some embodiments, BI₂ is about 4 weeks toabout 8 weeks. In some embodiments, BI₂ is about 6 weeks to about 8weeks. In some embodiments BI₂ is about 3 to about 4 weeks. In someembodiments, BI₂ is about 1 week. In some embodiments, BI₂ is about 2weeks. In some embodiments, BI₂ is about 3 weeks. In some embodiments,BI₂ is about 4 weeks. In some embodiments, BI₂ is about 5 weeks. In someembodiments, BI₂ is about 6 weeks. In some embodiments, BI₂ is about 7weeks. In some embodiments, BI₂ is about 8 weeks. In some embodiments,BI₂ is about 9 weeks. In some embodiments, BI₂ is about 10 weeks.

In some embodiments, the length of time between BD₃ and a subsequentdose that is part of the Booster Dosing Regimen, or between doses forany dose beyond BD₃ (BI_(n)) is each separately and independentlyselected from: about 1 week or more, about 2 weeks or more, or about 3weeks or more. In some embodiments, BI_(n) is about 1 week to about 12weeks. In some embodiments, BI_(n) is about 1 week to about 10 weeks. Insome embodiments, BI_(n) is about 2 weeks to about 10 weeks. In someembodiments, BI_(n) is about 2 weeks to about 8 weeks. In someembodiments, BI_(n) is about 3 weeks to about 8 weeks. In someembodiments, BI_(n) is about 4 weeks to about 8 weeks. In someembodiments, BI_(n) is about 6 weeks to about 8 weeks. In someembodiments BI_(n) is about 3 to about 4 weeks. In some embodiments,BI_(n) is about 1 week. In some embodiments, BI_(n) is about 2 weeks. Insome embodiments, BI_(n) is about 3 weeks. In some embodiments, BI_(n)is about 4 weeks. In some embodiments, BI_(n) is about 5 weeks. In someembodiments, BI_(n) is about 6 weeks. In some embodiments, BI_(n) isabout 7 weeks. In some embodiments, BI_(n) is about 8 weeks. In someembodiments, BI_(n) is about 9 weeks. In some embodiments, BI_(n) isabout 10 weeks.

In some embodiments, one or more compositions adminstered in BD₁ areformulated in a Tris buffer. In some embodiments, one or morecompositions administered in BD₂ are formulated in a Tris buffer. Insome embodiments, one or more compositions administering in BD₃ areformulated in a Tris buffer. In some embodiments, one or morecompositions administered in BD₃ are formulated in a Tris buffer.

In some embodiments, the Booster dosing regimen comprises administeringtwo or more mRNA compositions described herein, and at least two of themRNA compositions have different formulations. In some embodiments, theBooster dosing regimen comprises BD₁ and BD₂, where BD₁ comprisesadministering an mRNA formulated in a Tris buffer and BD₂ comprisesadministering an mRNA formulated in a PBS buffer. In some embodiments,the Booster dosing regimen comprises BD₁ and BD₂, where BD₁ comprisesadministering an mRNA formulated in a PBS buffer and BD₂ comprisesadministering an mRNA formulated in a Tris buffer.

In some embodiments, one or more mRNA compositions given in BD₁, BD₂,BD₃, and/or BD_(n) can be administered in combination with anothervaccine. In some embodiments, another vaccine is for a disease that isnot COVID-19. In some embodiments, the disease is one that increasesdeleterious effects of SARS-CoV-2 when a subject is coinfected with thedisease and SARS-CoV-2. In some embodiments, the disease is one thatincreases the transmission rate of SARS-CoV-2 when a subject iscoinfected with the disease and SARS-CoV-2. In some embodiments, anothervaccine is a different commerically available vaccine. In someembodiments, the different commercially available vaccine is an RNAvaccine. In some embodiments, the different commercially availablevaccine is a polypeptide-based vaccine. In some embodiments, anothervaccine (e.g., as described herein) and one or more mRNA compositionsgiven in BD₁, BD₂, BD₃, and/or BD_(n) are separately administered, forexample, in some embodiments via intramuscular injection, at differentinjection sites. For example, in some embodiments, an influenza vaccineand one or more SARS-CoV-2 mRNA compositions described herein given inBD₁, BD₂, BD₃, and/or BD_(n) are separately administered to differentarms of a subject via intramuscular injection.

Additional Booster Regimens

In some embodiments, methods of vaccination disclosed herein compriseadministering more than one Booster Dosing Regimen. In some embodiments,more than one Booster Dosing Regimen may need to be administered toincrease neutralizing antibody response. In some embodiments, more thanone booster dosing regimen may be needed to counteract a SARS-CoV-2strain that has been shown to have a high likelihood of evading immuneresponse elicited by vaccines that a patient has previously received. Insome embodiments, an additional Booster Dosing Regimen is administeredto a patient who has been determined to produce low concentrations ofneutralizing antibodies. In some embodiments, an additional boosterdosing regimen is administered to a patient who has been determined tohave a high likelihood of being susceptible to SARS-CoV-2 infection,despite previous vaccination (e.g., an immunocompromised patient, acancer patient, and/or an organ transplant patient).

The description provided above for the first Booster Dosing Regimen alsodescribes the one or more additional Booster Dosing Regimens. Theinterval of time between the first Booster Dosing Regimen and a secondBooster Dosing Regimen, or between subsequent Booster Dosing Regimenscan be any of the acceptable intervals of time described above betweenthe Primary Dosing Regimen and the First Booster Dosing Regimen.

In some embodiments, a dosing regimen comprises a primary regimen and abooster regimen, wherein at least one dose given in the primary regimenand/or the booster regimen comprises a composition comprising an RNAthat encodes a S protein or immunogenic fragment thereof from a variantthat is prevalent or is spreading rapidly in a relevant jurisdiction(e.g., Omicron variant as described herein). For example, in someembodiments, a primary regimen comprises at least 2 doses of BNT162b2(e.g., encoding a Wuhan strain), for example, given at least 3 weeksapart, and a booster regimen comprises at least 1 dose of a compositioncomprising RNA that encodes a S protein or immunogenic fragment thereoffrom a variant that is prevalent or is spreading rapidly in a relevantjurisdiction (e.g., Omicron variant as described herein). In some suchembodiments, such a dose of a booster regimen may further comprise anRNA that encodes a S protein or immunogenic fragment thereof from aWuhan strain, which can be administered with an RNA that encodes a Sprotein or immunogenic fragment thereof from a variant that is prevalentor is spreading rapidly in a relevant jurisdiction (e.g., Omicronvariant as described herein), as a single mixture, or as two separatecompositions, for example, in 1:1 weight ratio. In some embodiments, abooster regimen can also comprise at least 1 dose of BNT162b2, which canbe administered as a first booster dose or a subsequent booster dose.

In some embodiments, an RNA composition described herein is given as abooster at a dose that is higher than the doses given during a primaryregimen (primary doses) and/or the dose given for a first booster, ifany. For example, in some embodiments, such a dose may be 60 ug; or insome embodiments such a dose may be higher than 30 ug and lower than 60ug (e.g., 55 ug, 50 ug, or lower). In some embodiments, an RNAcomposition described herein is given as a booster at least 3-12 monthsor 4-12 months, or 5-12 months, or 6-12 months after the last dose(e.g., the last dose of a primary regimen or a first dose of a boosterregimen). In some embodiments, the primary doses and/or the firstbooster dose (if any) may comprise BNT162b2, for example at 30 ug perdose.

In some embodiments, an RNA composition described herein comprises anRNA encoding a polypeptide as set forth in SEQ ID NO: 49 or animmunogenic fragment thereof, or a variant thereof (e.g., having atleast 70% or more, including, e.g., at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or higher,identity to SEQ ID NO: 49). In some embodiments, an RNA compositioncomprises an RNA that includes the sequence of SEQ ID NO: 50 or avariant thereof (e.g., having at least 70% or more, including, e.g., atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or higher, identity to SEQ ID NO: 50). In someembodiments, an RNA composition comprises an RNA that includes thesequence of SEQ ID NO: 51 or a variant thereof (e.g., having at least70% or more, including, e.g., at least 80%, at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or higher,identity to SEQ ID NO: 51).

In some embodiments, an RNA composition described herein comprises anRNA encoding a polypeptide as set forth in SEQ ID NO: 55 or animmunogenic fragment thereof, or a variant thereof (e.g., having atleast 70% or more, including, e.g., at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or higher,identity to SEQ ID NO: 55. In some embodiments, an RNA compositioncomprises an RNA that includes the sequence of SEQ ID NO: 56 or avariant thereof (e.g., having at least 70% or more, including, e.g., atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or higher, identity to SEQ ID NO: 56). In someembodiments, an RNA composition comprises an RNA that includes thesequence of SEQ ID NO: 57 or a variant thereof (e.g., having at least70% or more, including, e.g., at least 80%, at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or higher,identity to SEQ ID NO: 57).

In some embodiments, an RNA composition described herein comprises anRNA encoding a polypeptide as set forth in SEQ ID NO: 58 or animmunogenic fragment thereof, or a variant thereof (e.g., having atleast 70% or more, including, e.g., at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or higher,identity to SEQ ID NO: 58). In some embodiments, an RNA compositioncomprises an RNA that includes the sequence of SEQ ID NO: 59 or avariant thereof (e.g., having at least 70% or more, including, e.g., atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or higher, identity to SEQ ID NO: 59). In someembodiments, an RNA composition comprises an RNA that includes thesequence of SEQ ID NO: 60 or a variant thereof (e.g., having at least70% or more, including, e.g., at least 80%, at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or higher,identity to SEQ ID NO: 60).

In some embodiments, an RNA composition described herein comprises anRNA encoding a polypeptide as set forth in SEQ ID NO: 61 or animmunogenic fragment thereof, or a variant thereof (e.g., having atleast 70% or more, including, e.g., at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or higher,identity to SEQ ID NO: 61). In some embodiments, an RNA compositioncomprises an RNA that includes the sequence of SEQ ID NO: 62a or avariant thereof (e.g., having at least 70% or more, including, e.g., atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or higher, identity to SEQ ID NO: 62a). In someembodiments, an RNA composition comprises an RNA that includes thesequence of SEQ ID NO: 63a or a variant thereof (e.g., having at least70% or more, including, e.g., at least 80%, at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or higher,identity to SEQ ID NO: 63a).

In some embodiments, the formulations disclosed herein can be used tocarry out any of the dosing regimens described in Table C (below).

TABLE C Exemplary Dosing Regimens: Primary Regimen Time between theBooster Regimen Time last dose of a Time Between Dose 1 and Primaryregimen Between Dose 1 and Dose 1 Dose 2 Doses 1 Dose 2 and a first doseof Dose 1 Dose 2 Doses 1 Dose 2 # (μg RNA) (μg RNA) and 2 FormulationBooster Regimen (μg RNA) (μg RNA) and 2 Formulation 1 30 30 2 to 8 weeksPBS At least 2 months 30 N/A¹ N/A PBS 2 30 30 2 to 8 weeks PBS At least3 months 30 N/A¹ N/A PBS 3 30 30 2 to 8 weeks PBS 6 to 12 months 30 N/A¹N/A PBS 4 30 30 2 to 8 weeks PBS or Tris 4 to 12 months 15 N/A¹ N/A PBSor Tris 5 30 30 2 to 8 weeks PBS or Tris 4 to 12 months 10 N/A¹ N/A PBSor Tris 6 30 30 2 to 8 weeks PBS or Tris 4 to 12 months 30 30 4 to 12PBS or Tris months 7 30 30 2 to 8 weeks PBS or Tris 4 to 12 months 30 154 to 12 PBS or Tris months 8 30 30 2 to 8 weeks PBS or Tris 4 to 12months 30 10 4 to 12 PBS or Tris months 9 30 30 2 to 8 weeks PBS or Tris4 to 12 months 30 60 4 to 12 PBS or Tris months 10 30 30 2 to 8 weeksPBS or Tris 4 to 12 months 30 >30 to <60 4 to 12 PBS or Tris months 1130 30 2 to 8 weeks PBS or Tris 4 to 12 months 30 50 4 to 12 PBS or Trismonths 12 30 30 2 to 8 weeks PBS At least 6 months 30 N/A¹ N/A PBS 13 3030 ~21 days PBS At least 2 months 30 N/A¹ N/A PBS 14 30 30 ~21 days PBSAt least 3 months 30 N/A¹ N/A PBS 15 30 30 ~21 days PBS 6 to 12 months30 N/A¹ N/A PBS 16 30 30 ~21 days PBS At least 6 months 30 N/A¹ N/A PBS17 30 30 21 days PBS At least 6 months 15 15 ~21 days PBS 18 30 30 21days PBS At least 6 months 15 15 ~21 days PBS 19 30 30 2 to 8 weeks PBSAt least 2 months 30 N/A¹ N/A Tris 20 30 30 2 to 8 weeks PBS At least 3months 30 N/A¹ N/A Tris 21 30 30 2 to 8 weeks PBS 6 to 12 months 30 N/A¹N/A Tris 22 30 30 2 to 8 weeks PBS At least 6 months 30 N/A¹ N/A Tris 2330 30 ~21 days PBS At least 2 months 30 N/A¹ N/A Tris 24 30 30 ~21 daysPBS At least 3 months 30 N/A¹ N/A Tris 25 30 30 ~21 days PBS 6 to 12months 30 N/A¹ N/A Tris 26 30 30 ~21 days PBS At least 6 months 30 N/A¹N/A Tris 27 30 30 21 days PBS At least 6 months 15 15 ~21 days Tris 2830 30 21 days PBS At least 6 months 15 15 ~21 days Tris 29 30 30 2 to 8weeks Tris At least 2 months 30 N/A¹ N/A Tris 30 30 30 2 to 8 weeks TrisAt least 3 months 30 N/A¹ N/A Tris 31 30 30 2 to 8 weeks Tris 6 to 12months 30 N/A¹ N/A Tris 32 30 30 2 to 8 weeks Tris At least 6 months 30N/A¹ N/A Tris 33 30 30 ~21 days Tris At least 2 months 30 N/A¹ N/A Tris34 30 30 ~21 days Tris At least 3 months 30 N/A¹ N/A Tris 35 30 30 ~21days Tris 6 to 12 months 30 N/A¹ N/A Tris 36 30 30 ~21 days Tris Atleast 6 months 30 N/A¹ N/A Tris 37 30 30 21 days Tris At least 6 months15 15 ~21 days Tris 38 30 30 21 days Tris At least 6 months 15 15 ~21days Tris 39 10 10 2 to 8 weeks Tris At least 2 months 10 N/A¹ N/A Tris40 10 10 2 to 8 weeks Tris At least 3 months 10 N/A¹ N/A Tris 41 10 10 2to 8 weeks Tris 6 to 12 months 10 N/A¹ N/A Tris 42 10 10 2 to 8 weeksTris At least 6 months 10 N/A¹ N/A Tris 43 10 10 ~21 days Tris At least2 months 10 N/A¹ N/A Tris 44 10 10 ~21 days Tris At least 3 months 10N/A¹ N/A Tris 45 10 10 ~21 days Tris 6 to 12 months 10 N/A¹ N/A Tris 4610 10 ~21 days Tris At least 6 months 10 N/A¹ N/A Tris 47 3 3 2 to 8weeks Tris At least 2 months 3 N/A¹ N/A Tris 48 3 3 2 to 8 weeks Tris Atleast 3 months 3 N/A¹ N/A Tris 49 3 3 2 to 8 weeks Tris 6 to 12 months 3N/A¹ N/A Tris 50 3 3 2 to 8 weeks Tris At least 6 months 3 N/A¹ N/A Tris51 3 3 ~21 days Tris At least 2 months 3 N/A¹ N/A Tris 52 3 3 ~21 daysTris At least 3 months 3 N/A¹ N/A Tris 53 3 3 ~21 days Tris 6 to 12months 3 N/A¹ N/A Tris 54 3 3 ~21 days Tris At least 6 months 3 N/A¹ N/ATris ¹N/A refers to no dose necessary.

In some embodiments of certain exemplary dosing regimens as described inTable C above, an RNA composition described herein (e.g., comprising RNAencoding a variant described herein) is given in a first dose of aprimary regimen. In some embodiments of certain exemplary dosingregimens as described in Table C above, an RNA composition describedherein (e.g., comprising RNA encoding a variant described herein) isgiven in a second dose of a primary regimen. In some embodiments ofcertain exemplary dosing regimens as described in Table C above, an RNAcomposition described herein (e.g., comprising RNA encoding a variantdescribed herein) is given in a first dose and a second dose of aprimary regimen. In some embodiments of certain exemplary dosingregimens as described in Table C above, an RNA composition describedherein (e.g., comprising RNA encoding a variant described herein) isgiven in a first dose of a booster regimen. In some embodiments ofcertain exemplary dosing regimens as described in Table C above, an RNAcomposition described herein (e.g., comprising RNA encoding a variantdescribed herein) is given in a second dose of a booster regimen. Insome embodiments of certain exemplary dosing regimens as described inTable C above, an RNA composition described herein (e.g., comprising RNAencoding a variant described herein) is given in a first dose and asecond dose of a booster regimen. In some embodiments of certainexemplary dosing regimens as described in Table C above, an RNAcomposition described herein (e.g., comprising RNA encoding a variantdescribed herein) is given in a first dose and a second dose of aprimary regimen and also in at least one dose of a booster regimen. Insome embodiments of certain exemplary dosing regimens as described inTable C above, an RNA composition described herein (e.g., comprising RNAencoding a variant described herein) is given in at least one dose(including, e.g., at least two doses) of a booster regimen and BNT162b2is given in a primary regimen. In some embodiments of certain exemplarydosing regimens as described in Table C above, an RNA compositiondescribed herein (e.g., comprising RNA encoding a variant describedherein) is given in a second dose of a booster regimen and BNT162b2 isgiven in a primary regimen and in a first dose of a booster regimen. Insome embodiments, an RNA composition described herein (e.g., comprisingRNA encoding a variant described herein) comprises an RNA encoding apolypeptide as set forth in SEQ ID NO: 49 or an immunogenic fragmentthereof, or a variant thereof (e.g., having at least 70% or more,including, e.g., at least 80%, at least 85%, at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, or higher, identity to SEQ IDNO: 49). In some embodiments, an RNA composition described herein (e.g.,comprising RNA encoding a variant described herein) comprises an RNAthat includes the sequence of SEQ ID NO: 50 or a variant thereof (e.g.,having at least 70% or more, including, e.g., at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, or higher, identity to SEQ ID NO: 50). In some embodiments, an RNAcomposition described herein (e.g., comprising RNA encoding a variantdescribed herein) comprises an RNA that includes the sequence of SEQ IDNO: 51 or a variant thereof (e.g., having at least 70% or more,including, e.g., at least 80%, at least 85%, at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, or higher, identity to SEQ IDNO: 51).

In some embodiments, an RNA composition described herein comprises anRNA encoding a polypeptide as set forth in SEQ ID NO: 55 or animmunogenic fragment thereof, or a variant thereof (e.g., having atleast 70% or more, including, e.g., at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or higher,identity to SEQ ID NO: 55). In some embodiments, an RNA compositioncomprises an RNA that includes the sequence of SEQ ID NO: 56 or avariant thereof (e.g., having at least 70% or more, including, e.g., atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or higher, identity to SEQ ID NO: 56). In someembodiments, an RNA composition comprises an RNA that includes thesequence of SEQ ID NO: 57 or a variant thereof (e.g., having at least70% or more, including, e.g., at least 80%, at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or higher,identity to SEQ ID NO: 57).

In some embodiments, an RNA composition described herein comprises anRNA encoding a polypeptide as set forth in SEQ ID NO: 58 or animmunogenic fragment thereof, or a variant thereof (e.g., having atleast 70% or more, including, e.g., at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or higher,identity to SEQ ID NO: 58). In some embodiments, an RNA compositioncomprises an RNA that includes the sequence of SEQ ID NO: 59 or avariant thereof (e.g., having at least 70% or more, including, e.g., atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or higher, identity to SEQ ID NO: 59). In someembodiments, an RNA composition comprises an RNA that includes thesequence of SEQ ID NO: 60 or a variant thereof (e.g., having at least70% or more, including, e.g., at least 80%, at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or higher,identity to SEQ ID NO: 60).

In some embodiments, an RNA composition described herein comprises anRNA encoding a polypeptide as set forth in SEQ ID NO: 61 or animmunogenic fragment thereof, or a variant thereof (e.g., having atleast 70% or more, including, e.g., at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or higher,identity to SEQ ID NO: 61). In some embodiments, an RNA compositioncomprises an RNA that includes the sequence of SEQ ID NO: 62a or avariant thereof (e.g., having at least 70% or more, including, e.g., atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or higher, identity to SEQ ID NO: 62a). In someembodiments, an RNA composition comprises an RNA that includes thesequence of SEQ ID NO: 63a or a variant thereof (e.g., having at least70% or more, including, e.g., at least 80%, at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or higher,identity to SEQ ID NO: 63a).

In some embodiments, such an RNA composition described herein (e.g.,comprising RNA encoding a variant described herein) can further compriseRNA encoding a S protein or an immunogenic fragment thereof of adifferent strain (e.g., a Wuhan strain). By way of example, in someembodiments, a second dose of a booster regimen of Regimens #9-11 asdescribed in Table C above can comprise an RNA composition describedherein (e.g., comprising RNA encoding a variant described herein such asOmicron, for example, in one embodiment RNA as described in thisExample) and a BNT162b2 construct, for example, in 1:1 weight ratio.

In some embodiments of Regimen #6 as described in Table C above, a firstdose and a second dose of a primary regimen and a first dose and asecond dose of a booster regimen each comprise an RNA compositiondescribed herein (e.g., comprising RNA encoding a variant describedherein such as Omicron, for example, in one embodiment RNA as describedin this Example). In some such embodiments, a second dose of a boosterregimen may not be necessary.

In some embodiments of Regimen #6 as described in Table C above, a firstdose and a second dose of a primary regimen and a first dose and asecond dose of a booster regimen each comprise an RNA compositiondescribed herein (e.g., comprising RNA encoding a variant describedherein such as Omicron, for example, in one embodiment RNA as describedin this Example). In some such embodiments, a second dose of a boosterregimen may not be necessary.

In some embodiments of Regimen #6 as described in Table C above, a firstdose and a second dose of a primary regimen each comprise a BNT162b2construct, and a first dose and a second dose of a booster regimen eachcomprise an RNA composition described herein (e.g., comprising RNAencoding a variant described herein such as Omicron, for example, in oneembodiment RNA as described in this Example). In some such embodiments,a second dose of a booster regimen may not be necessary.

In some embodiments of Regimen #6 as described in Table C above, a firstdose and a second dose of a primary regimen and a first dose of abooster regimen each comprise a BNT162b2 construct, and a second dose ofa booster regimen comprises an RNA composition described herein (e.g.,comprising RNA encoding a variant described herein such as Omicron, forexample, in one embodiment RNA as described in this Example).

Certain exemplary embodiments below are also within the scope of thepresent disclosure:

-   -   1. A composition or medical preparation comprising RNA        comprising a nucleotide sequence encoding a SARS-CoV-2 S protein        comprising one or more mutations characteristic of a SARS-CoV-2        variant (e.g., in some embodiments a SARS-CoV-2 Omicron        variant), or an immunogenic fragment thereof.    -   2. The composition or medical preparation of embodiment 1,        wherein the immunogenic fragment of the SARS-CoV-2 S protein        comprises the S1 subunit of the SARS-CoV-2 S protein, or the        receptor binding domain (RBD) of the S1 subunit of the        SARS-CoV-2 S protein.    -   3. The composition or medical preparation of embodiment 1 or 2,        wherein the SARS-CoV-2 S protein or immunogenic fragment thereof        comprises one or more mutations characteristic of a BA.1, BA.2,        BA.4/5, XBB, XBB.1, BQ.1.1, or a BA.4.6/BF.7 Omicron variant or        sublineages thereof.    -   4. The composition or medical preparation of embodiment 3,        wherein the SARS-CoV-2 S protein or immunogenic fragment thereof        comprises one or more mutations characteristic of a BA.4/5        variant, wherein the one or more mutations are selected from the        group consisting of T19I, Δ24-26, A27S, Δ69/70, G142D, V213G,        G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K,        L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G,        H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K.    -   5. The composition or medical preparation of embodiment 3,        wherein the SARS-CoV-2 S protein or immunogenic fragment thereof        comprises one or more mutations characteristic of a BA.1        variant, wherein the one or more mutations are selected from the        group consisting of A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211,        L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K,        G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H,        T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H,        N969K, L981F.    -   6. The composition or medical preparation of embodiment 3,        wherein the SARS-CoV-2 S protein or immunogenic fragment thereof        comprises one or more mutations characteristic of a BA.2        variant, wherein the one or more mutations are selected from the        group consisting of T19I, Δ24-26, A27S, G142D, V213G, G339D,        S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, S477N,        T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K,        P681H, N764K, D796Y, Q954H, and N969K.    -   7. The composition or medical preparation of embodiment 3,        wherein the SARS-CoV-2 S protein or immunogenic fragment thereof        comprises one or more mutations characteristic of a BA.2.75.1        variant, wherein the one or more mutations are selected from the        group consisting of T19I, Δ24-26, A27S, G142D, V213G, G339D,        S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, S477N,        T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K,        P681H, N764K, D796Y, Q954H, and N969K.    -   8. The composition or medical preparation of embodiment 3,        wherein the SARS-CoV-2 S protein or immunogenic fragment thereof        comprises one or more mutations characteristic of a BA.4.6/BF.7        variant, wherein the one or more mutations are selected from the        group consisting of T19I, Δ24-26, A27S, Δ69/70, G142D, V213G,        G339D, R346T, S371F, S373P, S375F, T376A, D405N, R408S, K417N,        N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H,        D614G, H655Y, N658S, N679K, P681H, N764K, D796Y, Q954H, and        N969K.    -   9. The composition or medical preparation of embodiment 3,        wherein the SARS-CoV-2 S protein or immunogenic fragment thereof        comprises one or more mutations characteristic of an XBB        variant, wherein the one or more mutations are selected from the        group consisting of T19I, Δ24-26, A27S, V83A, G142D, A144,        H146Q, Q183E, V213E, G339H, R346T, L368I, S371F, S373P, S375F,        T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N,        T478K, E484A, F486S, F490S, Q493R, Q498R, N501Y, Y505H, D614G,        H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K.    -   10. The composition or medical preparation of embodiment 3,        wherein the SARS-CoV-2 S protein or immunogenic fragment thereof        comprises one or more mutations characteristic of an XBB.1        variant, wherein the one or more mutations are selected from the        group consisting of T19I, Δ24-26, A27S, V83A, G142D, A144,        H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P,        S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K,        S477N, T478K, E484A, F486S, F490S, Q493R, Q498R, N501Y, Y505H,        D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K.    -   11. The composition or medical preparation of embodiment 3,        wherein the SARS-CoV-2 S protein or immunogenic fragment thereof        comprises one or more mutations characteristic of a BQ.1.1        variant, wherein the one or more mutations are selected from the        group consisting of T19I, Δ24-26, A27S, Δ69/70, G142D, V213G,        G339D, R346T, S371F, S373P, S375F, T376A, D405N, R408S, K417N,        N440K, K444T, L452R, N460K, S477N, T478K, E484A, F486V, Q498R,        N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H,        N969K.    -   12. The composition or medical preparation of any one of        embodiments 1-11, wherein the SARS-CoV-2 S protein comprising        one or more mutations characteristic of an Omicron variant, or        an immunogenic fragment thereof is encoded by a sequence that is        codon-optimized (e.g., codon-optimized for expression in human        cells) and/or which has a G/C content that is increased compared        to a wild type coding sequence.    -   13. The composition or medical preparation of embodiment 3 or 4,        wherein the SARS-CoV-2 S protein comprises one or more mutations        characteristic of an Omicron BA.4/5 variant and wherein:        -   a) the SARS-CoV-2 S protein comprises an amino acid sequence            having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%            identity to SEQ ID NO: 69, or an immunogenic fragment            thereof; and/or        -   b) the RNA encoding the SARS-CoV-2 S protein comprises a            nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%,            90%, 85%, or 80% identity to SEQ ID NO: 70; and/or at least            99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to SEQ ID            NO: 71.    -   14. The composition or medical preparation of embodiment 3 or 5,        wherein the SARS-CoV-2 S protein comprises one or more mutations        characteristic of an Omicron BA.1 variant and wherein:        -   a) the SARS-CoV-2 S protein comprises an amino acid sequence            having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%            identity to SEQ ID NO: 49, or an immunogenic fragment            thereof; and/or        -   b) the RNA encoding the SARS-CoV-2 S protein comprises a            nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%,            90%, 85%, or 80% identity to SEQ ID NO: 50 and/or at least            99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to SEQ ID            NO: 51.    -   15. The composition or medical preparation of embodiment 3 or 6,        wherein the SARS-CoV-2 S protein comprises one or more mutations        characteristic of an Omicron BA.2 variant and wherein:        -   a) the SARS-CoV-2 S protein comprises an amino acid sequence            having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%            identity to SEQ ID NO: 64, or an immunogenic fragment            thereof; and/or        -   b) the RNA encoding the SARS-CoV-2 S protein comprises a            nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%,            90%, 85%, or 80% identity to SEQ ID NO: 65 and/or at least            99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to SEQ ID            NO: 66.    -   16. The composition or medical preparation of embodiment 3 or 7,        wherein the SARS-CoV-2 S protein comprises one or more mutations        characteristic of an Omicron BA.2.75.1 variant and wherein:        -   a) the SARS-CoV-2 S protein comprises an amino acid sequence            having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%            identity to SEQ ID NO: 80, or an immunogenic fragment            thereof; and/or        -   b) the RNA encoding the SARS-CoV-2 S protein comprises a            nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%,            90%, 85%, or 80% identity to SEQ ID NO: 81 and/or at least            99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to SEQ ID            NO: 83.    -   17. The composition or medical preparation of embodiment 3 or 8,        wherein the SARS-CoV-2 S protein comprises one or more mutations        characteristic of an Omicron BA.4.6/BF.7 variant and wherein:        -   a) the SARS-CoV-2 S protein comprises an amino acid sequence            having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%            identity to SEQ ID NO: 90, or an immunogenic fragment            thereof; and/or        -   b) the RNA encoding the SARS-CoV-2 S protein comprises a            nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%,            90%, 85%, or 80% identity to SEQ ID NO: 91 and/or at least            99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to SEQ ID            NO: 92.    -   18. The composition or medical preparation of embodiment 3 or 9,        wherein the SARS-CoV-2 S protein comprises one or more mutations        characteristic of an Omicron XBB variant and wherein:        -   a) the SARS-CoV-2 S protein comprises an amino acid sequence            having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%            identity to SEQ ID NO: 95, or an immunogenic fragment            thereof; and/or        -   b) the RNA encoding the SARS-CoV-2 S protein comprises a            nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%,            90%, 85%, or 80% identity to SEQ ID NO: 96; and/or at least            99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to SEQ ID            NO: 98.    -   19. The composition or medical preparation of embodiment 3 or        11, wherein the SARS-CoV-2 S protein comprises one or more        mutations characteristic of an Omicron BQ.1.1 variant and        wherein:        -   a) the SARS-CoV-2 S protein comprises an amino acid sequence            having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%            identity to SEQ ID NO: 100, or an immunogenic fragment            thereof; and/or        -   b) the RNA encoding the SARS-CoV-2 S protein comprises a            nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%,            90%, 85%, or 80% identity to SEQ ID NO: 101 and/or at least            99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to SEQ ID            NO: 102.    -   20. The composition or medical preparation of any one of        embodiments 1-19, wherein the SARS-CoV-2 S protein comprises one        or more mutations that improves expression, stability, and/or        immunogenicity.    -   21. The composition or medical preparation of embodiment 20,        wherein the SARS-CoV-2 S protein comprises one or more mutations        that stabilizes the prefusion conformation.    -   22. The composition or medical preparation of embodiment 20,        wherein the SARS-CoV-2 S protein comprises proline mutations at        positions corresponding to residues 986 and 987 of SEQ ID NO: 1.    -   23. The composition or medical preparation of embodiment 21 or        22, wherein the SARS-CoV-2 S protein comprises one or more        proline residues at positions corresponding to positions 817,        892, 899, and/or 942 of SEQ ID NO: 1.    -   24. The composition or medical preparation of any one of        embodiments 20-23, wherein the SARS-CoV-2 S protein comprises a        mutation that prevents furin cleavage.    -   25. The composition or medical preparation of embodiment 24,        wherein the SARS-CoV-2 S protein comprises a mutation at a        location corresponding to residues 682-685 of SEQ ID NO: 1 that        prevents cleavage by a furin protease (e.g., a GSAS mutation).    -   26. The composition or medical preparation of any one of        embodiments 20-25, wherein the SARS-CoV-2 S protein comprises        one or more mutations that decreases S protein shedding (e.g.,        an aspartate to glycine mutation at a position corresponding to        residue 614 of SEQ ID NO: 1).    -   27. The composition or medical preparation of any one of        embodiments 1-26, wherein the RNA comprises a modified        nucleoside in place of uridine.    -   28. The composition or medical preparation of embodiment 27,        wherein the RNA comprises a modified nucleoside in place of each        uridine.    -   29. The composition or medical preparation of embodiment 27 or        28, wherein the modified nucleoside is selected from        pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and        5-methyl-uridine (m5U).    -   30. The composition or medical preparation of embodiment 29,        wherein the modified nucleoside is N1-methyl-pseudouridine        (m1ψ).    -   31. The composition or medical preparation of any one of        embodiments 1-30, wherein the RNA comprises a 5′ cap.    -   32. The composition or medical preparation of embodiment 31,        wherein the 5′ cap is or comprises a cap1 structure.    -   33. The composition or medical preparation of embodiment 32,        wherein the RNA comprises a 5′-cap that is or comprises m₂        ^(7,3′-O)Gppp(m₁ ^(2′-O))ApG.    -   34. The composition or medical preparation of any one of        embodiments 1-33, wherein the composition comprises a poly(A)        sequence.    -   35. The composition or medical preparation of embodiment 34,        wherein the poly(A) sequence comprises at least 100 A        nucleotides.    -   36. The composition or medical preparation of embodiment 34 or        35, wherein the poly(A) sequence is an interrupted sequence of A        nucleotides.    -   37. The composition or medical preparation of embodiment 36,        wherein the poly(A) sequence comprises 30 adenine nucleotides        followed by 70 adenine nucleotides, wherein the 30 adenine        nucleotides and 70 adenine nucleotides are separated by a linker        sequence.    -   38. The composition or medical preparation of embodiment 37,        wherein the poly(A) sequence comprises or consists of the        nucleotide sequence of SEQ ID NO: 14, or a nucleotide sequence        that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%        identical to the nucleotide sequence of SEQ ID NO: 14.    -   39. The composition or medical preparation of any one of        embodiments 1-38, wherein the RNA comprises a 5′-UTR that is or        comprises a modified human alpha-globin 5′-UTR.    -   40. The composition or medical preparation of embodiment 39,        wherein the 5′ UTR comprises the nucleotide sequence of SEQ ID        NO: 12, or a nucleotide sequence that is at least 99%, 98%, 97%,        96%, 95%, 90%, 85%, or 80% identical to the nucleotide sequence        of SEQ ID NO: 12.    -   41. The composition or medical preparation of any one of        embodiments 1-40, wherein the RNA comprises a 3′-UTR that is or        comprises a first sequence from the amino terminal enhancer of        split (AES) messenger RNA and a second sequence from the        mitochondrial encoded 12S ribosomal RNA.    -   42. The composition or medical preparation of embodiment 41,        wherein the RNA comprises a 3′ UTR comprising the nucleotide        sequence of SEQ ID NO: 13, or a nucleotide sequence having at        least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the        nucleotide sequence of SEQ ID NO: 13.    -   43. The composition or medical preparation of any one of        embodiments 1-42, wherein the RNA is formulated or is to be        formulated as particles.    -   44. The composition or medical preparation of embodiment 43,        wherein the particles are lipid nanoparticles (LNP) or lipoplex        (LPX) particles.    -   45. The composition or medical preparation of embodiment 44,        wherein the LNPs comprise a cationically ionizable lipid, a        neutral lipid, a sterol and a polymer-lipid conjugate.    -   46. The composition or medical preparation of embodiment 45,        wherein the neutral lipid is present in a concentration ranging        from about 5 to about 15 mol percent of the total lipids.    -   47. The composition or medical preparation of embodiment 45 or        46, wherein the cationically ionizable lipid is present in a        concentration ranging from about 40 to about 50 mol percent of        the total lipids.    -   48. The composition or medical preparation of any one of        embodiments 45-47, wherein the sterol is present in a        concentration ranging from about 30 to about 50 mol percent of        the total lipids.    -   49. The composition or medical preparation of any one of        embodiments 45-48, wherein the polymer-lipid conjugate is        present in a concentration ranging from about 1 to about 10 mol        percent of the total lipids.    -   50. The composition or medical preparation of any one of        embodiments 45-49, wherein the lipid nanoparticles comprise from        about 40 to about 50 mol percent of the cationically ionizable        lipid; from about 5 to about 15 mol percent of the neutral        lipid; from about 35 to about 45 mol percent of the sterol; and        from about 1 to about 10 mol percent of the polymer        conjugated-lipid.    -   51. The composition or medical preparation of embodiment 44,        wherein the RNA lipoplex particles are obtainable by mixing the        RNA with liposomes.    -   52. A composition or medical preparation comprising: a lipid        nanoparticle (LNP) comprising an RNA, wherein the RNA comprises        a nucleotide sequence that (a) encodes a SARS-CoV-2 S protein        comprising one or more mutations characteristic of an Omicron        BA.1 variant and comprising an amino acid sequence of SEQ ID NO:        49 or an amino acid sequence that is at least 80% (e.g., at 85%,        at least 90%, at least 91%, at least 92%, at least 93%, at least        94%, at least 95%, at least 97%, at least 98%, or 99% or higher)        identical to SEQ ID NO: 49 and/or (b) comprises the nucleotide        sequence of SEQ ID NO: 50 or a nucleotide sequence that is at        least 80% (e.g., at 85%, at least 90%, at least 91%, at least        92%, at least 93%, at least 94%, at least 95%, at least 97%, at        least 98%, or 99% or higher) identical to SEQ ID NO: 50, and        wherein the RNA comprises:        -   (a) modified uridines;        -   (b) a 5′ cap; and        -   wherein the LNP comprises a cationically ionizable lipid, a            neutral lipid, a sterol and a polymer-lipid conjugate.    -   53. The composition or medical preparation of embodiment 52,        wherein the nucleotide sequence includes modified uridines in        place of all uridines.    -   54. The composition or medical preparation of embodiment 52 or        53, wherein the modified uridines are each        N1-methyl-pseudouridine.    -   55. The composition or medical preparation of any one of        embodiments 52-54, wherein the RNA further comprises at least        one, at least two, or all of the following features:        -   a 5′ untranslated region (UTR) comprising SEQ ID NO: 12;        -   a 3′ untranslated region (UTR) comprising SEQ ID NO: 13; and        -   a poly-A sequence of at least 100 A nucleotides.    -   56. The composition or medical preparation of embodiment 55,        wherein the poly-A sequence comprises 30 adenine nucleotides        followed by 70 adenine nucleotides, wherein the 30 adenine        nucleotides and 70 adenine nucleotides are separated by a linker        sequence.    -   57. The composition or medical preparation of embodiment 56,        wherein the poly-A sequence comprises SEQ ID NO: 14.    -   58. The composition or medical preparation of any one of        embodiments 52-57, wherein the RNA comprises SEQ ID NO: 51.    -   59. A composition or medical preparation comprising a lipid        nanoparticle (LNP) comprising an RNA, wherein the RNA comprises        a nucleotide sequence (a) that encodes a SARS-CoV-2 S protein        comprising one or more mutations characteristic of a Beta        variant and comprising the amino acid sequence of SEQ ID NO: 55        or an amino acid sequence that is at least 80% (e.g., at 85%, at        least 90%, at least 91%, at least 92%, at least 93%, at least        94%, at least 95%, at least 97%, at least 98%, or 99% or higher)        identical to SEQ ID NO: 55 and/or (b) comprises the nucleotide        sequence of SEQ ID NO: 56 or a nucleotide sequence that is at        least 80% (e.g., at 85%, at least 90%, at least 91%, at least        92%, at least 93%, at least 94%, at least 95%, at least 97%, at        least 98%, or 99% or higher) identical to SEQ ID NO: 56, and        wherein the RNA comprises:        -   (a) modified uridines;        -   (b) a 5′ cap; and        -   wherein the LNP comprises a cationically ionizable lipid, a            neutral lipid, a sterol and a polymer-lipid conjugate.    -   60. The composition or medical preparation of embodiment 59,        wherein the nucleotide sequence includes modified uridines in        place of all uridines.    -   61. The composition or medical preparation of embodiment 59 or        60, wherein the modified uridines are each        N1-methyl-pseudouridine.    -   62. The composition or medical preparation of any one of        embodiments 59 to 61, wherein the RNA further comprises at least        one, at least two, or all of the following features:        -   a 5′ untranslated region (UTR) comprising SEQ ID NO: 12;        -   a 3′ untranslated region (UTR) comprising SEQ ID NO: 13; and            a poly-A sequence of at least 100 A nucleotides.    -   63. The composition or medical preparation of embodiment 62,        wherein the poly-A sequence comprises 30 adenine nucleotides        followed by 70 adenine nucleotides, wherein the 30 adenine        nucleotides and 70 adenine nucleotides are separated by a linker        sequence.    -   64. The composition or medical preparation of embodiment 63,        wherein the poly-A sequence comprises SEQ ID NO: 14.    -   65. The composition or medical preparation of any one of        embodiments 59-64, wherein the RNA comprises SEQ ID NO: 57.    -   66. A composition or medical preparation comprising a lipid        nanoparticle (LNP) comprising an RNA, wherein the RNA comprises        a nucleotide sequence that (a) encodes a SARS-CoV-2 S protein        comprising one or more mutations characteristic of an Alpha        variant and comprising the polypeptide of SEQ ID NO: 58 or an        amino acid sequence that is at least 80% (e.g., at 85%, at least        90%, at least 91%, at least 92%, at least 93%, at least 94%, at        least 95%, at least 97%, at least 98%, or 99% or higher)        identical to SEQ ID NO: 58 and/or (b) comprises the nucleotide        sequence of SEQ ID NO: 59 or a nucleotide sequence that is at        least 80% (e.g., at 85%, at least 90%, at least 91%, at least        92%, at least 93%, at least 94%, at least 95%, at least 97%, at        least 98%, or 99% or higher) identical to SEQ ID NO: 59, and        wherein the RNA comprises:        -   (a) modified uridines;        -   (b) a 5′ cap; and        -   wherein the LNP comprises a cationically ionizable lipid, a            neutral lipid, a sterol and a polymer-lipid conjugate.    -   67. The composition or medical preparation of embodiment 66,        wherein the nucleotide sequence includes modified uridines in        place of all uridines.    -   68. The composition or medical preparation of embodiment 66 or        67, wherein the modified uridines are each        N1-methyl-pseudouridine.    -   69. The composition or medical preparation of any one of        embodiments 66-68, wherein the RNA further comprises at least        one, at least two, or all of the following features:        -   a 5′ untranslated region (UTR) comprising SEQ ID NO: 12;        -   a 3′ untranslated region (UTR) comprising SEQ ID NO: 13; and        -   a poly-A sequence of at least 100 A nucleotides.    -   70. The composition or medical preparation of embodiment 69,        wherein the poly-A sequence comprises 30 adenine nucleotides        followed by 70 adenine nucleotides, wherein the 30 adenine        nucleotides and 70 adenine nucleotides are separated by a linker        sequence.    -   71. The composition or medical preparation of embodiment 70,        wherein the poly-A sequence comprises SEQ ID NO: 14.    -   72. The composition or medical preparation of any one of        embodiments 66-71, wherein the RNA comprises SEQ ID NO: 60.    -   73. A composition or medical preparation comprising a lipid        nanoparticle (LNP) comprising an RNA, wherein the RNA comprises        a nucleotide sequence that (a) encodes a SARS-CoV-2 S protein        comprising one or more mutations characteristic of a Delta        variant and comprises an amino acid sequence of SEQ ID NO: 61 or        an amino acid sequence that is at least 80% (e.g., at 85%, at        least 90%, at least 91%, at least 92%, at least 93%, at least        94%, at least 95%, at least 97%, at least 98%, or 99% or higher)        identical to SEQ ID NO: 61 and/or (b) comprises the nucleotide        sequence of SEQ ID NO: 62a or a nucleotide sequence that is at        least 80% (e.g., at 85%, at least 90%, at least 91%, at least        92%, at least 93%, at least 94%, at least 95%, at least 97%, at        least 98%, or 99% or higher) identical to SEQ ID NO: 62a, and        wherein the RNA comprises:        -   (a) modified uridines;        -   (b) a 5′ cap; and        -   wherein the LNP comprises a cationically ionizable lipid, a            neutral lipid, a sterol and a polymer-lipid conjugate.    -   74. The composition or medical preparation of embodiment 73,        wherein the nucleotide sequence includes modified uridines in        place of all uridines.    -   75. The composition or medical preparation of embodiment 73 or        74, wherein the modified uridines are each        N1-methyl-pseudouridine.    -   76. The composition or medical preparation of any one of        embodiments 73-75, wherein the RNA further comprises at least        one, at least two, or all of the following features:        -   a 5′ untranslated region (UTR) comprising SEQ ID NO: 12;        -   a 3′ untranslated region (UTR) comprising SEQ ID NO: 13; and            a poly-A sequence of at least 100 A nucleotides.    -   77. The composition or medical preparation of embodiment 76,        wherein the poly-A sequence comprises 30 adenine nucleotides        followed by 70 adenine nucleotides, wherein the 30 adenine        nucleotides and 70 adenine nucleotides are separated by a linker        sequence.    -   78. The composition or medical preparation of embodiment 77,        wherein the poly-A sequence comprises SEQ ID NO: 14.    -   79. The composition or medical preparation of any one of        embodiments 73-78, wherein the RNA comprises SEQ ID NO: 63a.    -   80. A composition or medical preparation comprising a lipid        nanoparticle (LNP) comprising an RNA, wherein the RNA comprises        a nucleotide sequence that (a) encodes a SARS-CoV-2 S protein        comprising one or more mutations characteristic of a BA.2        Omicron variant and encodes a polypeptide comprising an amino        acid sequence of SEQ ID NO: 64 or an amino acid sequence that is        at least 80% (e.g., at 85%, at least 90%, at least 91%, at least        92%, at least 93%, at least 94%, at least 95%, at least 97%, at        least 98%, or 99% or higher) identical to SEQ ID NO: 64        and/or (b) comprises the nucleotide sequence of SEQ ID NO: 65 or        a nucleotide sequence that is at least 80% (e.g., at 85%, at        least 90%, at least 91%, at least 92%, at least 93%, at least        94%, at least 95%, at least 97%, at least 98%, or 99% or higher)        identical to SEQ ID NO: 65, and wherein the RNA comprises:        -   (a) modified uridines;        -   (b) a 5′ cap; and        -   wherein the LNP comprises a cationically ionizable lipid, a            neutral lipid, a sterol and a polymer-lipid conjugate.    -   81. The composition or medical preparation of embodiment 80,        wherein the nucleotide sequence includes modified uridines in        place of all uridines.    -   82. The composition or medical preparation of embodiment 80 or        81, wherein the modified uridines are each        N1-methyl-pseudouridine.    -   83. The composition or medical preparation of any one of        embodiments 80-82, wherein the RNA further comprises at least        one, at least two, or all of the following features:        -   a 5′ untranslated region (UTR) comprising SEQ ID NO: 12;        -   a 3′ untranslated region (UTR) comprising SEQ ID NO: 13; and            a poly-A sequence of at least 100 A nucleotides.    -   84. The composition or medical preparation of embodiment 83,        wherein the poly-A sequence comprises 30 adenine nucleotides        followed by 70 adenine nucleotides, wherein the 30 adenine        nucleotides and 70 adenine nucleotides are separated by a linker        sequence.    -   85. The composition or medical preparation of embodiment 84,        wherein the poly-A sequence comprises SEQ ID NO: 14.    -   86. The composition or medical preparation of any one of        embodiments 80-85, wherein the RNA comprises SEQ ID NO: 67.    -   87. A composition or medical preparation comprising a lipid        nanoparticle (LNP) comprising an RNA, wherein the RNA comprises        a nucleotide sequence that (a) encodes a SARS-CoV-2 S protein        that comprises one or more mutations characteristic of a BA.2.75        Omicron variant and comprises an amino acid sequence of SEQ ID        NO 80 or an amino acid sequence that is at least 80% (e.g., at        85%, at least 90%, at least 91%, at least 92%, at least 93%, at        least 94%, at least 95%, at least 97%, at least 98%, or 99% or        higher) identical to SEQ ID NO: 80 and/or (b) comprises the        nucleotide sequence of SEQ ID NO: 81 or a nucleotide sequence        that is at least 80% (e.g., at 85%, at least 90%, at least 91%,        at least 92%, at least 93%, at least 94%, at least 95%, at least        97%, at least 98%, or 99% or higher) identical to SEQ ID NO: 81,        and wherein the RNA comprises:        -   (a) modified uridines;        -   (b) a 5′ cap; and        -   wherein the LNP comprises a cationically ionizable lipid, a            neutral lipid, a sterol and a polymer-lipid conjugate.    -   88. The composition or medical preparation of embodiment 87,        wherein the nucleotide sequence includes modified uridines in        place of all uridines.    -   89. The composition or medical preparation of embodiment 87 or        88, wherein the modified uridines are each        N1-methyl-pseudouridine.    -   90. The composition or medical preparation of any one of        embodiments 87-89, wherein the RNA further comprises at least        one, at least two, or all of the following features:        -   a 5′ untranslated region (UTR) comprising SEQ ID NO: 12;        -   a 3′ untranslated region (UTR) comprising SEQ ID NO: 13; and            a poly-A sequence of at least 100 A nucleotides.    -   91. The composition or medical preparation of embodiment 90,        wherein the poly-A sequence comprises 30 adenine nucleotides        followed by 70 adenine nucleotides, wherein the 30 adenine        nucleotides and 70 adenine nucleotides are separated by a linker        sequence.    -   92. The composition or medical preparation of embodiment 91,        wherein the poly-A sequence comprises SEQ ID NO: 14.    -   93. The composition or medical preparation of any one of        embodiments 90-92, wherein the RNA comprises SEQ ID NO: 83.    -   94. A composition or medical preparation comprising a lipid        nanoparticle (LNP) comprising an RNA, wherein the RNA comprises        a nucleotide sequence that encodes a SARS-CoV-2 S protein        comprising one or more mutations characteristic of a BA.2.75.2        Omicron variant and comprises an amino acid sequence of SEQ ID        NO 85 or an amino acid sequence that is at least 80% (e.g., at        85%, at least 90%, at least 91%, at least 92%, at least 93%, at        least 94%, at least 95%, at least 97%, at least 98%, or 99% or        higher) identical to SEQ ID NO: 85 and/or (b) comprises the        nucleotide sequence of 86 or a nucleotide sequence that is at        least 80% (e.g., at 85%, at least 90%, at least 91%, at least        92%, at least 93%, at least 94%, at least 95%, at least 97%, at        least 98%, or 99% or higher) identical to SEQ ID NO: 86, and        wherein the RNA comprises:        -   (a) modified uridines;        -   (b) a 5′ cap; and        -   wherein the LNP comprises a cationically ionizable lipid, a            neutral lipid, a sterol and a polymer-lipid conjugate.    -   95. The composition or medical preparation of embodiment 94,        wherein the nucleotide sequence includes modified uridines in        place of all uridines.    -   96. The composition or medical preparation of embodiment 94 or        95, wherein the modified uridines are each        N1-methyl-pseudouridine.    -   97. The composition or medical preparation of any one of        embodiments 94-96, wherein the RNA further comprises at least        one, at least two, or all of the following features:        -   a 5′ untranslated region (UTR) comprising SEQ ID NO: 12;        -   a 3′ untranslated region (UTR) comprising SEQ ID NO: 13; and            a poly-A sequence of at least 100 A nucleotides.    -   98. The composition or medical preparation of embodiment 97,        wherein the poly-A sequence comprises 30 adenine nucleotides        followed by 70 adenine nucleotides, wherein the 30 adenine        nucleotides and 70 adenine nucleotides are separated by a linker        sequence.    -   99. The composition or medical preparation of embodiment 98,        wherein the poly-A sequence comprises SEQ ID NO: 14.    -   100. The composition or medical preparation of any one of        embodiments 94-99, wherein the RNA comprises SEQ ID NO: 88.    -   101. A composition or medical preparation comprising a lipid        nanoparticle (LNP) comprising an RNA, wherein the RNA comprises        a nucleotide sequence that (a) encodes a SARS-CoV-2 S protein        comprising one or more mutations characteristic of an Omicron        BA.4/5 variant and comprises an amino acid sequence of SEQ ID        NO: 69 or an amino acid sequence that is at least 80% (e.g., at        85%, at least 90%, at least 91%, at least 92%, at least 93%, at        least 94%, at least 95%, at least 97%, at least 98%, or 99% or        higher) identical to SEQ ID NO: 69 and/or (b) comprises the        nucleotide sequence of SEQ ID NO: 70 or a nucleotide sequence        that is at least 80% (e.g., at 85%, at least 90%, at least 91%,        at least 92%, at least 93%, at least 94%, at least 95%, at least        97%, at least 98%, or 99% or higher) identical to SEQ ID NO: 70,        and wherein the RNA comprises:        -   (a) modified uridines;        -   (b) a 5′ cap; and        -   wherein the LNP comprises a cationically ionizable lipid, a            neutral lipid, a sterol and a polymer-lipid conjugate.    -   102. The composition or medical preparation of embodiment 101,        wherein the nucleotide sequence includes modified uridines in        place of all uridines.    -   103. The composition or medical preparation of embodiment 101 or        102, wherein the modified uridines are each        N1-methyl-pseudouridine.    -   104. The composition or medical preparation of any one of        embodiments 101-103, wherein the RNA further comprises at least        one, at least two, or all of the following features:        -   a 5′ untranslated region (UTR) comprising SEQ ID NO: 12;        -   a 3′ untranslated region (UTR) comprising SEQ ID NO: 13; and            a poly-A sequence of at least 100 A nucleotides.    -   105. The composition or medical preparation of embodiment 104,        wherein the poly-A sequence comprises 30 adenine nucleotides        followed by 70 adenine nucleotides, wherein the 30 adenine        nucleotides and 70 adenine nucleotides are separated by a linker        sequence.    -   106. The composition or medical preparation of embodiment 105,        wherein the poly-A sequence comprises SEQ ID NO: 14.    -   107. The composition or medical preparation of any one of        embodiments 101-106, wherein the RNA comprises SEQ ID NO: 72.    -   108. A composition or medical preparation comprising a lipid        nanoparticle (LNP) comprising an RNA, wherein the RNA comprises        a nucleotide sequence that (a) encodes a SARS-CoV-2 S protein        comprising one or more mutations that are characteristic of an        Omicron BA.4.6/BF.7 variant and comprising an amino acid        sequence of SEQ ID NO: 90 or an amino acid sequence that is at        least 80% (e.g., at 85%, at least 90%, at least 91%, at least        92%, at least 93%, at least 94%, at least 95%, at least 97%, at        least 98%, or 99% or higher) identical to SEQ ID NO: 90 and (b)        comprises the nucleotide sequence of SEQ ID NO: 91 or a        nucleotide sequence that is at least 80% (e.g., at 85%, at least        90%, at least 91%, at least 92%, at least 93%, at least 94%, at        least 95%, at least 97%, at least 98%, or 99% or higher)        identical to SEQ ID NO: 91, and wherein the RNA comprises:        -   (a) modified uridines;        -   (b) a 5′ cap; and        -   wherein the LNP comprises a cationically ionizable lipid, a            neutral lipid, a sterol and a polymer-lipid conjugate.    -   109. The composition or medical preparation of embodiment 108,        wherein the nucleotide sequence includes modified uridines in        place of all uridines.    -   110. The composition or medical preparation of embodiment 108 or        109, wherein the modified uridines are each        N1-methyl-pseudouridine.    -   111. The composition or medical preparation of any one of        embodiments 108-110, wherein the RNA further comprises at least        one, at least two, or all of the following features:        -   a 5′ untranslated region (UTR) comprising SEQ ID NO: 12;        -   a 3′ untranslated region (UTR) comprising SEQ ID NO: 13; and            a poly-A sequence of at least 100 A nucleotides.    -   112. The composition or medical preparation of embodiment 111,        wherein the poly-A sequence comprises 30 adenine nucleotides        followed by 70 adenine nucleotides, wherein the 30 adenine        nucleotides and 70 adenine nucleotides are separated by a linker        sequence.    -   113. The composition or medical preparation of embodiment 112,        wherein the poly-A sequence comprises SEQ ID NO: 14.    -   114. The composition or medical preparation of any one of        embodiments 108-112, wherein the RNA comprises SEQ ID NO: 93.    -   115. A composition or medical preparation comprising a lipid        nanoparticle (LNP) comprising an RNA, wherein the RNA comprises        a nucleotide sequence that (a) encodes a SARS-CoV-2 S protein        comprising one or more mutations characteristic of an XBB        Omicron variant and comprises an amino acid sequence of SEQ ID        NO 85 or an amino acid sequence that is at least 80% (e.g., at        85%, at least 90%, at least 91%, at least 92%, at least 93%, at        least 94%, at least 95%, at least 97%, at least 98%, or 99% or        higher) identical to SEQ ID NO: 95 and/or (b) comprises the        nucleotide sequence of SEQ ID NO: 96 or a nucleotide sequence        that is at least 80% (e.g., at 85%, at least 90%, at least 91%,        at least 92%, at least 93%, at least 94%, at least 95%, at least        97%, at least 98%, or 99% or higher) identical to SEQ ID NO: 96,        and wherein the RNA comprises:        -   (a) modified uridines;        -   (b) a 5′ cap; and        -   wherein the LNP comprises a cationically ionizable lipid, a            neutral lipid, a sterol and a polymer-lipid conjugate.    -   116. The composition or medical preparation of embodiment 115,        wherein the nucleotide sequence includes modified uridines in        place of all uridines.    -   117. The composition or medical preparation of embodiment 115 or        116, wherein the modified uridines are each        N1-methyl-pseudouridine.    -   118. The composition or medical preparation of any one of        embodiments 115-117, wherein the RNA further comprises at least        one, at least two, or all of the following features:        -   a 5′ untranslated region (UTR) comprising SEQ ID NO: 12;        -   a 3′ untranslated region (UTR) comprising SEQ ID NO: 13; and            a poly-A sequence of at least 100 A nucleotides.    -   119. The composition or medical preparation of embodiment 118,        wherein the poly-A sequence comprises 30 adenine nucleotides        followed by 70 adenine nucleotides, wherein the 30 adenine        nucleotides and 70 adenine nucleotides are separated by a linker        sequence.    -   120. The composition or medical preparation of embodiment 119,        wherein the poly-A sequence comprises SEQ ID NO: 14.    -   121. The composition or medical preparation of any one of        embodiments 115-120, wherein the RNA comprises SEQ ID NO: 98.    -   122. A composition or medical preparation comprising a lipid        nanoparticle (LNP) comprising an RNA, wherein the RNA comprises        a nucleotide sequence that (a) encodes a SARS-CoV-2 S protein        comprising one or more mutations characteristic of a BQ.1.1        Omicron variant and comprising an amino acid sequence of SEQ ID        NO 100 or an amino acid sequence that is at least 80% (e.g., at        85%, at least 90%, at least 91%, at least 92%, at least 93%, at        least 94%, at least 95%, at least 97%, at least 98%, or 99% or        higher) identical to SEQ ID NO: 100 and/or (b) comprises a        nucleotide sequence of SEQ ID NO: 101 or a nucleotide sequence        that is at least 80% (e.g., at 85%, at least 90%, at least 91%,        at least 92%, at least 93%, at least 94%, at least 95%, at least        97%, at least 98%, or 99% or higher) identical to SEQ ID NO:        101, and wherein the RNA comprises:        -   (a) modified uridines;        -   (b) a 5′ cap; and        -   wherein the LNP comprises a cationically ionizable lipid, a            neutral lipid, a sterol and a polymer-lipid conjugate.    -   123. The composition or medical preparation of embodiment 122,        wherein the nucleotide sequence includes modified uridines in        place of all uridines.    -   124. The composition or medical preparation of embodiment 122 or        123, wherein the modified uridines are each        N1-methyl-pseudouridine.    -   125. The composition or medical preparation of any one of        embodiments 122-124, wherein the RNA further comprises at least        one, at least two, or all of the following features:        -   a 5′ untranslated region (UTR) comprising SEQ ID NO: 12;        -   a 3′ untranslated region (UTR) comprising SEQ ID NO: 13; and            a poly-A sequence of at least 100 A nucleotides.    -   126. The composition or medical preparation of embodiment 125,        wherein the poly-A sequence comprises 30 adenine nucleotides        followed by 70 adenine nucleotides, wherein the 30 adenine        nucleotides and 70 adenine nucleotides are separated by a linker        sequence.    -   127. The composition or medical preparation of embodiment 126,        wherein the poly-A sequence comprises SEQ ID NO: 14.    -   128. The composition or medical preparation of any one of        embodiments 121-127, wherein the RNA comprises SEQ ID NO: 103.    -   129. The composition or medical preparation of any one of        embodiments 1-128, wherein the RNA is present in an amount        within a range of about 1 μg to about 100 μg per dose in the        composition.    -   130. The composition or medical preparation of embodiment 129,        wherein the RNA is present in an amount within a range of about        1 μg to about 60 μg per dose in the composition.    -   131. The composition or medical preparation of embodiment 130,        wherein the RNA is present in an amount of about 1.5 μg, about        2.5 μg, about 3.0 μg, about 5.0 μg, about 10 μg, about 15 μg,        about 30 μg, or about 60 μg per dose in the composition.    -   132. The composition or medical preparation of any one of        embodiments 1-131, further comprising a second RNA comprising a        nucleotide sequence encoding a SARS-CoV-2 S protein, or an        immunogenic fragment thereof, wherein the SARS-CoV-2 S protein        comprises one or more mutations that are characteristic of a        second SARS-CoV-2 variant.    -   133. A composition or medical preparation comprising:        -   (a) a first RNA comprising a nucleotide sequence encoding a            SARS-CoV-2 S protein of a first strain or variant, or an            immunogenic fragment thereof; and        -   (b) a second RNA comprising a nucleotide sequence encoding a            SARS-CoV-2 S protein of a second variant, or an immunogenic            fragment thereof,        -   wherein the first variant is different from the second            variant, and optionally wherein the first and/or second            variant is an Omicron variant.    -   134. The composition or medical preparation of embodiment 133,        wherein the first RNA comprises a nucleotide sequence that        encodes a SARS-CoV-2 S protein of a Wuhan strain, or an        immunogenic fragment thereof, and the second RNA comprises a        nucleotide sequence that encodes a SARS-CoV-2 S protein        comprising one or more mutations that are characteristic of an        Omicron variant, or an immunogenic fragment thereof.    -   135. The composition or medical preparation of embodiments 133        or 134, wherein the first RNA comprises a nucleotide sequence        that encodes a SARS-CoV-2 S protein of a Wuhan strain, or an        immunogenic fragment thereof, and the second RNA comprises a        nucleotide sequence that encodes a SARS-CoV-2 S protein        comprising one or more mutations that are characteristic of a        BA.4/5, BA.1, BA.2, XBB, XBB.1, or BQ.1.1 Omicron variant, or        sublineages thereof or an immunogenic fragment thereof.    -   136. The composition or medical preparation of embodiment 133,        wherein the second RNA comprises a nucleotide sequence that        encodes a SARS-CoV-2 S protein that is antigenically distinct        from the S protein encoded by the first RNA, or an immunogenic        fragment thereof.    -   137. A composition or medical preparation comprising (a) a first        RNA comprising a nucleotide sequence encoding a SARS-CoV-2 S        protein of a Wuhan strain, an Alpha variant, a Beta variant, a        Delta variant, or a BA.1 Omicron variant, or an immunogenic        fragment thereof and (b) a second RNA comprising a nucleotide        sequence encoding a SARS-CoV-2 S protein comprising one or more        mutations that are characteristic of an Omicron variant that is        not a BA.1 Omicron variant, or an immunogenic fragment thereof.    -   138. The composition or medical preparation of any one of        embodiments 133-137, wherein the second RNA comprises a        nucleotide sequence that encodes a SARS-CoV-2 S protein        comprising one or more mutations characteristic of a BA.4/5        Omicron variant, or a variant that has evolved from a BA.4/5        Omicron variant, or an immunogenic fragment thereof.    -   139. The composition or medical preparation of any one of        embodiments 133-135, wherein the first RNA comprises a        nucleotide sequence encoding a SARS-CoV-2 S protein of a Wuhan        strain, or an immunogenic fragment thereof, and the second RNA        comprises a nucleotide sequence that encodes a SARS-CoV-2 S        protein comprising one or more mutations that are characteristic        of a BA.1 Omicron variant, or an immunogenic fragment thereof.    -   140. The composition or medical preparation of any one of        embodiments 133-138, wherein the first RNA comprises a        nucleotide sequence that encodes a SARS-CoV-2 S protein of a        Wuhan strain, or an immunogenic fragment thereof, and the second        RNA comprises a nucleotide sequence that encodes a SARS-CoV-2 S        protein comprising one or more mutations characteristic of a        BA.2 Omicron variant, or an immunogenic fragment thereof.    -   141. The composition or medical preparation of any one of        embodiments 133-138, wherein the first RNA comprises a        nucleotide sequence that encodes a SARS-CoV-2 S protein of a        Wuhan strain, or an immunogenic fragment thereof, and the second        RNA comprises a nucleotide sequence that encodes a SARS-CoV-2 S        protein comprising one or more mutations characteristic of a        BA.4 or BA.5 Omicron variant, or an immunogenic fragment        thereof.    -   142. The composition or medical preparation of any one of        embodiments 133-138, wherein the first RNA comprises a        nucleotide sequence encoding a SARS-CoV-2 S protein of a Wuhan        strain, or an immunogenic fragment thereof, and the second RNA        comprises a nucleotide sequence encoding a SARS-CoV-2 S protein        comprising one or more mutations characteristic of a BA.2        Omicron variant, or an immunogenic fragment thereof.    -   143. The composition or medical preparation of any one of        embodiments 133-138, wherein the first RNA comprises a        nucleotide sequence encoding a SARS-CoV-2 S protein of a Wuhan        strain, or an immunogenic fragment thereof, and the second RNA        comprises a nucleotide sequence encoding a SARS-CoV-2 S protein        comprising one or more mutations characteristic of a BA.4 or        BA.5 Omicron variant, or an immunogenic fragment thereof.    -   144. The composition or medical preparation of any one of        embodiments 133-143, wherein the first RNA comprises a        nucleotide sequence encoding a SARS-CoV-2 S protein of a Wuhan        strain, or an immunogenic fragment thereof, and the second RNA        comprises a nucleotide sequence encoding a SARS-CoV-2 S protein        comprising one or more mutations characteristic of an XBB        Omicron variant, or an immunogenic fragment thereof.    -   145. The composition or medical preparation of any one of        embodiments 133-138, wherein the first RNA comprises a        nucleotide sequence encoding a SARS-CoV-2 S protein of a Wuhan        strain, or an immunogenic fragment thereof, and the second RNA        comprises a nucleotide sequence encoding a SARS-CoV-2 S protein        comprising one or more mutations characteristic of a BQ.1.1        Omicron variant or sublineages thereof, or an immunogenic        fragment thereof.    -   146. The composition or medical preparation of anyone of        embodiments 133-145, wherein the immunogenic fragment of the        SARS-CoV-2 S protein encoded by the first RNA and/or the second        RNA comprises the S1 subunit of the SARS-CoV-2 S protein, or the        receptor binding domain (RBD) of the S1 subunit of the        SARS-CoV-2 S protein.    -   147. The composition or medical preparation of any one of        embodiments 133-146, wherein each of the first RNA and the        second RNA is codon-optimized (e.g., codon-optimized for        expression in human cells) and/or has a G/C content that is        increased compared to a wild type coding sequence.    -   148. A composition or medical preparation comprising a first RNA        and a second RNA, wherein:        -   a) the first RNA comprises a nucleotide sequence encoding a            SARS-CoV-2 S protein of a Wuhan strain wherein (i) the            SARS-CoV-2 S protein of a Wuhan strain comprises SEQ ID NO:            7 or an amino acid sequence that is at least 80% (e.g., at            85%, at least 90%, at least 91%, at least 92%, at least 93%,            at least 94%, at least 95%, at least 97%, at least 98%, or            99% or higher) identical to SEQ ID NO: 7, and/or (ii) the            first RNA comprises a nucleotide sequence of SEQ ID NO: 9 or            20 or a nucleotide sequence that is at least 80% (e.g., at            85%, at least 90%, at least 91%, at least 92%, at least 93%,            at least 94%, at least 95%, at least 97%, at least 98%, or            99% or higher) identical to SEQ ID NO: 9 or 20, and        -   b) the second RNA comprises a nucleotide sequence encoding a            SARS-CoV-2 protein comprising one or more mutations            characteristic of a BA.4/5 Omicron variant, wherein (i) the            SARS-CoV-2 S protein comprising one or more mutations            characteristic of a BA.4/5 Omicron variant comprises an            amino acid sequence of 90 or an amino acid sequence that is            at least 80% (e.g., at 85%, at least 90%, at least 91%, at            least 92%, at least 93%, at least 94%, at least 95%, at            least 97%, at least 98%, or 99% or higher) identical to SEQ            ID NO: 90 and/or (ii) the second RNA comprises the            nucleotide sequence of SEQ ID NO: 91 or a nucleotide            sequence that is at least 80% (e.g., at 85%, at least 90%,            at least 91%, at least 92%, at least 93%, at least 94%, at            least 95%, at least 97%, at least 98%, or 99% or higher)            identical to SEQ ID NO: 91.    -   149. A composition or medical preparation comprising a first RNA        and a second RNA, wherein:        -   a) the first RNA comprises a nucleotide sequence encoding a            SARS-CoV-2 S protein of a Wuhan strain wherein (i) the            SARS-CoV-2 S protein of a Wuhan strain comprises SEQ ID NO:            7 or an amino acid sequence that is at least 80% (e.g., at            85%, at least 90%, at least 91%, at least 92%, at least 93%,            at least 94%, at least 95%, at least 97%, at least 98%, or            99% or higher) identical to SEQ ID NO: 7, and/or (ii) the            first RNA comprises a nucleotide sequence of SEQ ID NO: 9 or            20 or a nucleotide sequence that is at least 80% (e.g., at            85%, at least 90%, at least 91%, at least 92%, at least 93%,            at least 94%, at least 95%, at least 97%, at least 98%, or            99% or higher) identical to SEQ ID NO: 9 or 20, and        -   b) the second RNA comprises a nucleotide sequence encoding a            SARS-CoV-2 protein comprising one or more mutations            characteristic of a BA.1 Omicron variant, wherein (i) the            SARS-CoV-2 S protein comprising one or more mutations            characteristic of a BA.1 Omicron variant comprises an amino            acid sequence of SEQ ID NO: 49 or an amino acid sequence            that is at least 80% (e.g., at 85%, at least 90%, at least            91%, at least 92%, at least 93%, at least 94%, at least 95%,            at least 97%, at least 98%, or 99% or higher) identical to            SEQ ID NO: 49 and/or (ii) the second RNA comprises the            nucleotide sequence of SEQ ID NO: 70 or a nucleotide            sequence that is at least 80% (e.g., at 85%, at least 90%,            at least 91%, at least 92%, at least 93%, at least 94%, at            least 95%, at least 97%, at least 98%, or 99% or higher)            identical to SEQ ID NO: 70.    -   150. A composition or medical preparation comprising a first RNA        and a second RNA, wherein:        -   a) the first RNA comprises a nucleotide sequence encoding a            SARS-CoV-2 S protein of a Wuhan strain wherein (i) the            SARS-CoV-2 S protein of a Wuhan strain comprises SEQ ID NO:            7 or an amino acid sequence that is at least 80% (e.g., at            85%, at least 90%, at least 91%, at least 92%, at least 93%,            at least 94%, at least 95%, at least 97%, at least 98%, or            99% or higher) identical to SEQ ID NO: 7, and/or (ii) the            first RNA comprises a nucleotide sequence of SEQ ID NO: 9 or            20 or a nucleotide sequence that is at least 80% (e.g., at            85%, at least 90%, at least 91%, at least 92%, at least 93%,            at least 94%, at least 95%, at least 97%, at least 98%, or            99% or higher) identical to SEQ ID NO: 9 or 20, and        -   b) the second RNA comprises a nucleotide sequence encoding a            SARS-CoV-2 protein comprising one or more mutations            characteristic of a BA.2 Omicron variant, wherein (i) the            SARS-CoV-2 S protein comprising one or more mutations            characteristic of a BA.2 Omicron variant comprises an amino            acid sequence of SEQ ID NO: 64 or an amino acid sequence            that is at least 80% (e.g., at 85%, at least 90%, at least            91%, at least 92%, at least 93%, at least 94%, at least 95%,            at least 97%, at least 98%, or 99% or higher) identical to            SEQ ID NO: 69 and/or (ii) the second RNA comprises the            nucleotide sequence of SEQ ID NO: 70 or a nucleotide            sequence that is at least 80% (e.g., at 85%, at least 90%,            at least 91%, at least 92%, at least 93%, at least 94%, at            least 95%, at least 97%, at least 98%, or 99% or higher)            identical to SEQ ID NO: 70.    -   151. A composition or medical preparation comprising a first RNA        and a second RNA, wherein:        -   a) the first RNA comprises a nucleotide sequence encoding a            SARS-CoV-2 S protein of a Wuhan strain wherein (i) the            SARS-CoV-2 S protein of a Wuhan strain comprises SEQ ID NO:            7 or an amino acid sequence that is at least 80% (e.g., at            85%, at least 90%, at least 91%, at least 92%, at least 93%,            at least 94%, at least 95%, at least 97%, at least 98%, or            99% or higher) identical to SEQ ID NO: 7, and/or (ii) the            first RNA comprises a nucleotide sequence of SEQ ID NO: 9 or            20 or a nucleotide sequence that is at least 80% (e.g., at            85%, at least 90%, at least 91%, at least 92%, at least 93%,            at least 94%, at least 95%, at least 97%, at least 98%, or            99% or higher) identical to SEQ ID NO: 9 or 20, and        -   b) the second RNA comprises a nucleotide sequence encoding a            SARS-CoV-2 protein comprising one or more mutations            characteristic of a BA.2.75 Omicron variant, wherein (i) the            SARS-CoV-2 S protein comprising one or more mutations            characteristic of a BA.2.75 Omicron variant comprises an            amino acid sequence of SEQ ID NO: 80 or an amino acid            sequence that is at least 80% (e.g., at 85%, at least 90%,            at least 91%, at least 92%, at least 93%, at least 94%, at            least 95%, at least 97%, at least 98%, or 99% or higher)            identical to SEQ ID NO: 80 and/or (ii) the second RNA            comprises the nucleotide sequence of SEQ ID NO: 81 or a            nucleotide sequence that is at least 80% (e.g., at 85%, at            least 90%, at least 91%, at least 92%, at least 93%, at            least 94%, at least 95%, at least 97%, at least 98%, or 99%            or higher) identical to SEQ ID NO: 81.    -   152. A composition or medical preparation comprising a first RNA        and a second RNA, wherein:        -   a) the first RNA comprises a nucleotide sequence encoding a            SARS-CoV-2 S protein of a Wuhan strain wherein (i) the            SARS-CoV-2 S protein of a Wuhan strain comprises SEQ ID NO:            7 or an amino acid sequence that is at least 80% (e.g., at            85%, at least 90%, at least 91%, at least 92%, at least 93%,            at least 94%, at least 95%, at least 97%, at least 98%, or            99% or higher) identical to SEQ ID NO: 7, and/or (ii) the            first RNA comprises a nucleotide sequence of SEQ ID NO: 9 or            20 or a nucleotide sequence that is at least 80% (e.g., at            85%, at least 90%, at least 91%, at least 92%, at least 93%,            at least 94%, at least 95%, at least 97%, at least 98%, or            99% or higher) identical to SEQ ID NO: 9 or 20, and        -   b) the second RNA comprises a nucleotide sequence encoding a            SARS-CoV-2 protein comprising one or more mutations            characteristic of a BA.2.75.2 Omicron variant, wherein (i)            the SARS-CoV-2 S protein comprising one or more mutations            characteristic of a BA.2.75.2 Omicron variant comprises an            amino acid sequence of SEQ ID NO: 85 or an amino acid            sequence that is at least 80% (e.g., at 85%, at least 90%,            at least 91%, at least 92%, at least 93%, at least 94%, at            least 95%, at least 97%, at least 98%, or 99% or higher)            identical to SEQ ID NO: 85 and/or (ii) the second RNA            comprises the nucleotide sequence of SEQ ID NO: 86 or a            nucleotide sequence that is at least 80% (e.g., at 85%, at            least 90%, at least 91%, at least 92%, at least 93%, at            least 94%, at least 95%, at least 97%, at least 98%, or 99%            or higher) identical to SEQ ID NO: 86.    -   153. A composition or medical preparation comprising a first RNA        and a second RNA, wherein:        -   a) the first RNA comprises a nucleotide sequence encoding a            SARS-CoV-2 S protein of a Wuhan strain wherein (i) the            SARS-CoV-2 S protein of a Wuhan strain comprises SEQ ID NO:            7 or an amino acid sequence that is at least 80% (e.g., at            85%, at least 90%, at least 91%, at least 92%, at least 93%,            at least 94%, at least 95%, at least 97%, at least 98%, or            99% or higher) identical to SEQ ID NO: 7, and/or (ii) the            first RNA comprises a nucleotide sequence of SEQ ID NO: 9 or            20 or a nucleotide sequence that is at least 80% (e.g., at            85%, at least 90%, at least 91%, at least 92%, at least 93%,            at least 94%, at least 95%, at least 97%, at least 98%, or            99% or higher) identical to SEQ ID NO: 9 or 20, and        -   b) the second RNA comprises a nucleotide sequence encoding a            SARS-CoV-2 protein comprising one or more mutations            characteristic of a BA.4.6 or BF.7 Omicron variant,            wherein (i) the SARS-CoV-2 S protein comprising one or more            mutations characteristic of a BA.4.6 or BF.7 Omicron variant            comprises an amino acid sequence of SEQ ID NO: 90 or an            amino acid sequence that is at least 80% (e.g., at 85%, at            least 90%, at least 91%, at least 92%, at least 93%, at            least 94%, at least 95%, at least 97%, at least 98%, or 99%            or higher) identical to SEQ ID NO: 90 and/or (ii) the second            RNA comprises the nucleotide sequence of SEQ ID NO: 91 or a            nucleotide sequence that is at least 80% (e.g., at 85%, at            least 90%, at least 91%, at least 92%, at least 93%, at            least 94%, at least 95%, at least 97%, at least 98%, or 99%            or higher) identical to SEQ ID NO: 91.    -   154. A composition or medical preparation comprising a first RNA        and a second RNA, wherein:        -   a) the first RNA comprises a nucleotide sequence encoding a            SARS-CoV-2 S protein of a Wuhan strain wherein (i) the            SARS-CoV-2 S protein of a Wuhan strain comprises SEQ ID NO:            7 or an amino acid sequence that is at least 80% (e.g., at            85%, at least 90%, at least 91%, at least 92%, at least 93%,            at least 94%, at least 95%, at least 97%, at least 98%, or            99% or higher) identical to SEQ ID NO: 7, and/or (ii) the            first RNA comprises a nucleotide sequence of SEQ ID NO: 9 or            20 or a nucleotide sequence that is at least 80% (e.g., at            85%, at least 90%, at least 91%, at least 92%, at least 93%,            at least 94%, at least 95%, at least 97%, at least 98%, or            99% or higher) identical to SEQ ID NO: 9 or 20, and        -   b) the second RNA comprises a nucleotide sequence encoding a            SARS-CoV-2 protein comprising one or more mutations            characteristic of an XBB Omicron variant, wherein (i) the            SARS-CoV-2 S protein comprising one or more mutations            characteristic of an XBB Omicron variant comprises an amino            acid sequence of SEQ ID NO: 95 or an amino acid sequence            that is at least 80% (e.g., at 85%, at least 90%, at least            91%, at least 92%, at least 93%, at least 94%, at least 95%,            at least 97%, at least 98%, or 99% or higher) identical to            SEQ ID NO: 95 and/or (ii) the second RNA comprises the            nucleotide sequence of SEQ ID NO: 96 or a nucleotide            sequence that is at least 80% (e.g., at 85%, at least 90%,            at least 91%, at least 92%, at least 93%, at least 94%, at            least 95%, at least 97%, at least 98%, or 99% or higher)            identical to SEQ ID NO: 96.    -   155. A composition or medical preparation comprising a first RNA        and a second RNA, wherein:        -   a) the first RNA comprises a nucleotide sequence encoding a            SARS-CoV-2 S protein of a Wuhan strain wherein (i) the            SARS-CoV-2 S protein of a Wuhan strain comprises SEQ ID NO:            7 or an amino acid sequence that is at least 80% (e.g., at            85%, at least 90%, at least 91%, at least 92%, at least 93%,            at least 94%, at least 95%, at least 97%, at least 98%, or            99% or higher) identical to SEQ ID NO: 7, and/or (ii) the            first RNA comprises a nucleotide sequence of SEQ ID NO: 9 or            20 or a nucleotide sequence that is at least 80% (e.g., at            85%, at least 90%, at least 91%, at least 92%, at least 93%,            at least 94%, at least 95%, at least 97%, at least 98%, or            99% or higher) identical to SEQ ID NO: 9 or 20, and        -   b) the second RNA comprises a nucleotide sequence encoding a            SARS-CoV-2 protein comprising one or more mutations            characteristic of an BQ.1.1 Omicron variant, wherein (i) the            SARS-CoV-2 S protein comprising one or more mutations            characteristic of a BQ.1.1 Omicron variant comprises an            amino acid sequence of SEQ ID NO: 100 or an amino acid            sequence that is at least 80% (e.g., at 85%, at least 90%,            at least 91%, at least 92%, at least 93%, at least 94%, at            least 95%, at least 97%, at least 98%, or 99% or higher)            identical to SEQ ID NO: 100 and/or (ii) the second RNA            comprises the nucleotide sequence of SEQ ID NO: 100 or a            nucleotide sequence that is at least 80% (e.g., at 85%, at            least 90%, at least 91%, at least 92%, at least 93%, at            least 94%, at least 95%, at least 97%, at least 98%, or 99%            or higher) identical to SEQ ID NO: 100.    -   156. The composition or medical preparation of any one of        embodiments 133-155, wherein each of the first RNA and the        second RNA encode a SARS-CoV-2 S protein comprising one or more        mutations that improves expression, stability, and/or        immunogenicity.    -   157. The composition or medical preparation of embodiment 156,        wherein each of the first RNA and the second RNA encode a        SARS-CoV-2 S protein comprising one or more mutations that        stabilize the prefusion confirmation.    -   158. The composition or medical preparation of embodiment 157,        wherein each of the first RNA and the second RNA encode a        SARS-CoV-2 S protein comprising proline mutations at positions        corresponding to residues 986 and 987 of SEQ ID NO: 1.    -   159. The composition or medical preparation of embodiment 157 or        158, wherein each of the first RNA and the second RNA encode a        SARS-CoV-2 S protein comprising one or more proline mutations at        positions corresponding to residues 817, 892, 899, and/or 942 of        SEQ ID NO: 1.    -   160. The composition or medical preparation of any one of        embodiments 156-159, wherein each of the first RNA and the        second RNA encode a SARS-CoV-2 S protein comprising a mutation        that prevents furin cleavage.    -   161. The composition or medical preparation of embodiment 160,        wherein each of the first RNA and the second RNA encode a        SARS-CoV-2 S protein comprising a mutation at a location        corresponding to residues 682-685 of SEQ ID NO: 1 that prevents        cleavage by a furin protease (e.g., a GSAS mutation).    -   162. The composition or medical preparation of any one of        embodiments 156-161, wherein each of the first RNA and the        second RNA encode a SARS-CoV-2 S protein comprising one or more        mutations that decreases S protein shedding (e.g., an aspartate        to glycine mutation at a position corresponding to residue 614        of SEQ ID NO: 1).    -   163. The composition or medical preparation of any one of        embodiments 133-162, wherein the first RNA and the second RNA        each comprise a modified nucleoside in place of uridine.    -   164. The composition or medical preparation of embodiment 163,        wherein the first RNA and the second RNA each comprise a        modified nucleoside in place of each uridine.    -   165. The composition or medical preparation of embodiment 163 or        164, wherein the modified nucleoside is selected from        pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and        5-methyl-uridine (m5U).    -   166. The composition or medical preparation of embodiment 165,        wherein the modified nucleoside is N1-methyl-pseudouridine        (m1ψ).    -   167. The composition or medical preparation of any one of        embodiments 133-166, wherein the first RNA and the second RNA        each comprise a 5′ cap.    -   168. The composition or medical preparation of embodiment 167,        wherein the 5′ cap is or comprises a cap1 structure.    -   169. The composition or medical preparation of embodiment 168,        wherein the 5′-cap is or comprises m₂ ^(7,3′-O)Gppp(m₁        ^(2′-O))ApG.    -   170. The composition or medical preparation or medical        preparation of any one of embodiments 133-169, wherein the first        RNA and the second RNA each comprise a poly(A) sequence.    -   171. The composition or medical preparation of embodiment 170,        wherein the poly(A) sequence comprises at least 100 A        nucleotides.    -   172. The composition or medical preparation of embodiment 170 or        171, wherein the poly(A) sequence is an interrupted sequence of        A nucleotides.    -   173. The composition or medical preparation of embodiment 172,        wherein the poly(A) sequence comprises 30 adenine nucleotides        followed by 70 adenine nucleotides, wherein the adenine        nucleotides and 70 adenine nucleotides are separated by a linker        sequence.    -   174. The composition or medical preparation of any one of        embodiments 170-173, wherein the poly(A) sequence comprises or        consists of the nucleotide sequence of SEQ ID NO: 14, or a        nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%,        90%, 85%, or 80% identical to the nucleotide sequence of SEQ ID        NO: 14.    -   175. The composition or medical preparation of any one of        embodiments 133-174, wherein the first RNA and the second RNA        each comprise a 5′-UTR that is or comprises a modified human        alpha-globin 5′-UTR.    -   176. The composition or medical preparation of embodiment 175,        wherein the 5′ UTR comprises the nucleotide sequence of SEQ ID        NO: 12, or a nucleotide sequence that is at least 99%, 98%, 97%,        96%, 95%, 90%, 85%, or 80% identical to the nucleotide sequence        of SEQ ID NO: 12.    -   178. The composition or medical preparation of any one of        embodiments 133-176, wherein the first RNA and the second RNA        each comprise a 3′-UTR that is or comprises a first sequence        from the amino terminal enhancer of split (AES) messenger RNA        and a second sequence from the mitochondrial encoded 12S        ribosomal RNA.    -   179. The composition or medical preparation of embodiment 178,        wherein the first RNA and the second RNA each comprise a 3′ UTR        comprises the nucleotide sequence of SEQ ID NO: 13, or a        nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%,        90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID        NO: 13.    -   180. The composition or medical preparation of any one of        embodiments 133-179, wherein the first RNA and the second RNA        are each formulated or are to be formulated as particles.    -   181. The composition or medical preparation of embodiment 180,        wherein the particles are lipid nanoparticles (LNP) or lipoplex        (LPX) particles.    -   182. The composition or medical preparation of embodiment 181,        wherein the LNPs comprise a cationically ionizable lipid, a        neutral lipid, a sterol and a polymer-lipid conjugate.    -   183. The composition or medical preparation of embodiment 181 or        182, wherein the first RNA and the second RNA are formulated in        separate lipid nanoparticles.    -   184. The composition or medical preparation of embodiment 181 or        182, wherein the first RNA and the second RNA are formulated in        the same lipid nanoparticles.    -   185. The composition or medical preparation of any one of        embodiments 182-184, wherein the neutral lipid is present in a        concentration ranging from about 5 to about 15 mol percent of        the total lipids.    -   186. The composition or medical preparation of any one of        embodiments 182-185, wherein the cationically ionizable lipid is        present in a concentration ranging from about 40 to about 50 mol        percent of the total lipids.    -   187. The composition or medical preparation of any one of        embodiments 182-186, wherein the sterol is present in a        concentration ranging from about 30 to about 50 mol percent of        the total lipids.    -   188. The composition or medical preparation of any one of        embodiments 182-187, wherein the polymer-lipid conjugate is        present in a concentration ranging from about 1 to about 10 mol        percent of the total lipids.    -   189. The composition or medical preparation of any one of        embodiments 182-188, wherein the lipid nanoparticles comprise        from about 40 to about 50 mol percent of the cationically        ionizable lipid; from about 5 to about 15 mol percent of the        neutral lipid; from about 35 to about 45 mol percent of the        sterol; and from about 1 to about 10 mol percent of the polymer        conjugated-lipid.    -   190. The composition or medical preparation of embodiment 181,        wherein the RNA lipoplex particles are obtainable by mixing the        first RNA and the second RNA with liposomes.    -   191. The composition or medical preparation of any one of        embodiments 133-190, wherein the first RNA and the second RNA        are present in a combined amount within a range of about 1 μg to        about 100 μg per dose.    -   192. The composition or medical preparation of any one of        embodiments 133-191, wherein the first RNA and the second RNA        are present in a combined amount within a range of about 1 μg to        about 60 μg per dose.    -   193. The composition or medical preparation of any one of        embodiments 133-192, wherein the first RNA and the second RNA        are present in a combined amount of about 3.0 μg, about 10 μg,        about 30 μg, or about 60 μg per dose.    -   194. The composition or medical preparation of any one of        embodiments 133-193, wherein the ratio of the first RNA to the        second RNA is about 1:10 to about 10:1.    -   195. The composition or medical preparation of any one of        embodiments 133-194, wherein the ratio of the first RNA to the        second RNA is about 1:1.    -   196. The composition or medical preparation of any one of        embodiments 133-195, wherein:        -   a) the first RNA and the second RNA are each present in an            amount of about 1.5 μg per dose in the composition;        -   b) the first RNA and the second RNA are each present in an            amount of about 5 μg per dose in the composition;        -   c) the first RNA and the second RNA are each present in an            amount of about 15 μg per dose in the composition; or        -   d) the first RNA and the second RNA are each present in an            amount of about 15 μg per dose in the composition.    -   197. The composition or medical preparation of any one of        embodiments 124-169, further comprising an RNA encoding one or        more T cell epitopes of SARS-CoV-2.    -   198. The composition or medical preparation of embodiment 170,        wherein the RNA encoding one or more T cell epitopes of        SARS-CoV-2 comprises one or more epitopes of each of the ORF1ab,        M, and N regions of the SARS-CoV-2 genome.    -   199. The composition or medical preparation of any one of        embodiments 1-198, which is formulated or is to be formulated as        a liquid, a solid, or a combination thereof.    -   200. The composition or medical preparation of any one of        embodiments 1-199, which is formulated or is to be formulated        for injection.    -   201. The composition or medical preparation of any one of        embodiments 1-200, wherein the composition or medical        preparation is formulated or is to be formulated for        intramuscular administration.    -   202. The composition or medical preparation of any one of        embodiments 1-201, which is a pharmaceutical composition.    -   203. The composition or medical preparation of any one of        embodiments 1-202, which is a vaccine.    -   204. The composition or medical preparation of embodiment 202 or        203, wherein the pharmaceutical composition further comprises        one or more pharmaceutically acceptable carriers, diluents        and/or excipients.    -   205. The composition or medical preparation of any one of        embodiments 1-204, which is a kit.    -   206. The composition or medical preparation of embodiment 205,        further comprising instructions for use of the composition or        medical preparation for inducing an immune response against        coronavirus in a subject.    -   207. The composition or medical preparation of any one of        embodiments 1-206 for pharmaceutical use.    -   208. The composition or medical preparation of embodiment 207,        wherein the pharmaceutical use comprises inducing an immune        response against coronavirus in a subject.    -   209. The composition or medical preparation of embodiment 207 or        208, wherein the pharmaceutical use comprises a therapeutic or        prophylactic treatment of a coronavirus infection.    -   210. The composition or medical preparation of embodiment 208 or        209, wherein the coronavirus is a sarbecovirus.    -   211. The composition or medical preparation of any one of        embodiments 208-211, wherein the coronavirus is a        betacoronavirus.    -   212. The composition or medical preparation of any one of        embodiments 208-212, wherein the coronavirus is SARS-CoV-2.    -   213. The composition or medical preparation of any one of        embodiments 1-212, which is for administration to a human.    -   214. A method of eliciting an immune response against SARS-CoV-2        in a subject comprising administering the composition or medical        preparation of any one of embodiments 1-213.    -   215. The method of embodiment 214, wherein the immune response        is elicited against an Omicron variant of SARS-CoV-2.    -   216. The method of embodiment 214 or 215, wherein the subject        has previously been infected with or vaccinated against        SARS-CoV-2.    -   217. The method of any one of embodiments 214-216, wherein an        antigen of a Wuhan strain of SARS-CoV-2 has previously been        delivered to the subject (e.g., as a polypeptide or an RNA        encoding such a polypeptide).    -   218. The method of any one of embodiments 214-217, wherein the        subject has previously been administered RNA encoding a        SARS-CoV-2 S protein of a Wuhan strain.    -   219. The method of any one of embodiments 214-218, wherein the        subject has previously been administered two or more doses of        RNA encoding a SARS-CoV-2 S protein of a Wuhan strain.    -   220. The method of embodiment 219, wherein the RNA of any one of        embodiments 1-206 is administered at least about 2 months after        the two or more doses of RNA encoding a SARS-CoV-2 S protein of        a Wuhan strain.    -   221. The method of any one of embodiments 214-220, wherein the        method comprises administering RNA encoding an antigen of a        SARS-CoV-2 virus that is not a BA.1 Omicron variant.    -   222. The method of any one of embodiments 214-221, wherein the        method comprises administering RNA comprising a nucleotide        sequence encoding a SARS-Cov-2 S protein comprising one or more        mutations characteristic of an Omicron variant, wherein the        Omicron variant is not a BA.1 Omicron variant.    -   223. The method of any one of embodiments 214-222, wherein the        method comprises administering RNA comprising a nucleotide        sequence encoding a SARS-CoV-2 S protein comprising one or more        mutations characteristic of a BA.2 Omicron variant.    -   224. The method of any one of embodiments 214-222, wherein the        method comprises administering RNA comprising a nucleotide        sequence encoding a SARS-CoV-2 S protein comprising one or more        mutations characteristic of a BA.4 or BA.5 Omicron variant.    -   225. A method for inducing an immune response in a subject,        wherein the method comprises administering (a) a first RNA        comprising a nucleotide sequence encoding a SARS-CoV-2 S protein        of a Wuhan strain, or comprising one or more mutations        characteristic of an Alpha variant, a Beta variant, or a Delta        variant, and (b) a second RNA comprising a nucleotide sequence        encoding a SARS-CoV-2 S protein comprising one or more mutations        characteristic of an Omicron variant that is not a BA.1 Omicron        variant.    -   226. The method of embodiment 225, where the second RNA        comprises a nucleotide sequence encoding a SARS-CoV-2 S protein        of a BA.2 Omicron variant.    -   227. The method of embodiment 225, where the second RNA        comprises a nucleotide sequence encoding an S protein comprising        one or more mutations characteristic of a BA.4 or BA.5 Omicron        variant.    -   228. A method for inducing an immune response in a subject,        wherein the method comprises administering (a) a first RNA        comprising a nucleotide sequence encoding a SARS-CoV-2 S protein        of a Wuhan strain, or comprising one or more mutations        characteristic of an Alpha variant, a Beta variant, a Delta        variant, or a BA.1 Omicron variant and (b) a second RNA        comprising a nucleotide sequence encoding a SARS-CoV-2 S protein        that is antigenically distinct from the S protein encoded by the        first RNA.    -   229. The method of embodiment 228, wherein the second RNA        comprises a nucleotide sequence encoding a SARS-CoV-2 S protein        comprising one or more mutations that are characteristic of an        Omicron variant that is not a BA.1 Omicron variant.    -   230. The method of embodiment 228, wherein the second RNA        comprises a nucleotide sequence encoding a SARS-CoV-2 S protein        comprising one or more mutations that are characteristic of a        BA.2 Omicron variant.    -   231. The method of embodiment 228, wherein the second RNA        comprises a nucleotide sequence encoding a SARS-CoV-2 S protein        comprising one or more mutations characteristic of a BA.4 or        BA.5 Omicron variant.    -   232. The method of any one of embodiments 214-231, wherein the        first RNA and the second RNA are encapsulated in separate LNPs.    -   233. The method of any one of embodiments 214-231, wherein the        first RNA and the second RNA are encapsulated in the same LNP.    -   234. The method of any one of embodiments 214-232, wherein the        first RNA and the second RNA are administered separately, e.g.,        at different injection sites.    -   235. The method of any one of embodiments 214-234, further        comprising administering an RNA comprising a nucleotide sequence        encoding one or more T cell epitopes of SARS-CoV-2.    -   236. The method of embodiment 235, wherein the nucleotide        sequence encoding one or more T cell epitopes of SARS-CoV-2        encodes one or more epitopes of each of the ORF1ab, M, and N        regions of the SARS-CoV-2 genome.    -   237. The method of embodiment 235 or 236, wherein the first RNA,        the second RNA, and the RNA encoding one or more T cell epitopes        of SARS-CoV-2 are each formulated in separate LNPs.    -   238. The method of embodiment 235 or 236, wherein the first RNA        and the second RNA are co-formulated in the same LNP, and the        RNA encoding one or more T cell epitopes of SARS-CoV-2 is        formulated in a separate LNP.    -   239. The method of embodiment 235 or 236, wherein each of the        first RNA, the second RNA, and the RNA encoding one or more T        cell epitopes of SARS-CoV-2 is co-formulated in the same LNP.    -   240. The method of any one of embodiments 235-239, wherein the        first RNA, the second RNA, and the RNA encoding one or more T        cell epitopes of SARS-CoV-2 are administered in a dose        comprising:        -   (a) 30 μg of the first RNA and the second RNA combined            (e.g., 15 μg of the first RNA and 15 μg of the second RNA),            and 5 μg of the RNA encoding one or more T cell epitopes of            SARS-CoV-2;        -   (b) 30 μg of the first RNA and the second RNA combined            (e.g., 15 μg of the first RNA and 15 μg of the second RNA),            and 10 μg of the RNA encoding one or more T cell epitopes of            SARS-CoV-2; or        -   (c) 30 μg of the first RNA and the second RNA combined            (e.g., 15 μg of the first RNA and 15 μg of the second RNA),            and 15 μg of the RNA encoding one or more T cell epitopes of            SARS-CoV-2.    -   241. The method of any one of embodiments 235-240, wherein the        RNA encoding one or more T-cell epitopes of SARS-CoV-2 encodes a        polypeptide sequence of SEQ ID NO: RS C7p2full.    -   242. The method of any one of embodiments 207-241, further        comprising administering one or more vaccines against a        respiratory disease.    -   243. The method of embodiment 242, wherein the respiratory        disease is RSV or influenza.    -   244. The method of embodiment 242 or 243, comprising        administering a vaccine against RSV and a vaccine against        influenza.    -   245. The method of any one of embodiments 242-244, wherein the        one or more vaccines against a respiratory disease are RNA        vaccines each comprise one or more RNAs encoding an antigenic        protein.    -   246. The method of embodiment 245, comprising administering one        or more RNAs encoding antigen(s) from an influenza virus and/or        one or more RNAs encoding antigen(s) from an RSV.    -   247. The method of embodiment 246, wherein each RNA is        formulated in a separate LNP.    -   248. The method of embodiment 246, wherein each RNA encoding a        SARS-CoV-2 S protein or an immunogenic fragment thereof is        co-formulated together in the same LNP, each RNA encoding an        antigenic protein from an influenza virus is co-formulated        together in the same LNP, and each RNA encoding an antigenic        protein from an RSV is co-formulated together in the same LNP.    -   249. The method of embodiment 246, wherein each RNA encoding a        SARS-CoV-2 S protein or an immunogenic fragment thereof, each        RNA encoding an antigenic protein from an influenza virus, and        each RNA encoding an antigenic protein from an RSV are        co-formulated together in the same LNP.    -   250. A composition or medical preparation comprising RNA        encoding an amino acid sequence comprising a SARS-CoV-2 S        protein, an immunogenic variant thereof, or an immunogenic        fragment of the SARS-CoV-2 S protein or the immunogenic variant        thereof.    -   251. The composition or medical preparation of embodiment 250,        wherein an immunogenic fragment of the SARS-CoV-2 S protein        comprises the S1 subunit of the SARS-CoV-2 S protein, or the        receptor binding domain (RBD) of the S1 subunit of the        SARS-CoV-2 S protein.    -   252. The composition or medical preparation of embodiments 250        or 251, wherein the amino acid sequence comprising a SARS-CoV-2        S protein, an immunogenic variant thereof, or an immunogenic        fragment of the SARS-CoV-2 S protein or the immunogenic variant        thereof is encoded by a coding sequence which is codon-optimized        and/or the G/C content of which is increased compared to wild        type coding sequence, wherein the codon-optimization and/or the        increase in the G/C content preferably does not change the        sequence of the encoded amino acid sequence.    -   253. The composition or medical preparation of any one of        embodiments 250 to 252, wherein    -   (i) the RNA encoding a SARS-CoV-2 S protein, an immunogenic        variant thereof, or an immunogenic fragment of the SARS-CoV-2 S        protein or the immunogenic variant thereof comprises the        nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2,        8 or 9, a nucleotide sequence having at least 99%, 98%, 97%,        96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence        of nucleotides 979 to 1584 of SEQ ID NO: 2, 8 or 9, or a        fragment of the nucleotide sequence of nucleotides 979 to 1584        of SEQ ID NO: 2, 8 or 9, or the nucleotide sequence having at        least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the        nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2,        8 or 9; and/or    -   (ii) a SARS-CoV-2 S protein, an immunogenic variant thereof, or        an immunogenic fragment of the SARS-CoV-2 S protein or the        immunogenic variant thereof comprises the amino acid sequence of        amino acids 327 to 528 of SEQ ID NO: 1, an amino acid sequence        having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%        identity to the amino acid sequence of amino acids 327 to 528 of        SEQ ID NO: 1, or an immunogenic fragment of the amino acid        sequence of amino acids 327 to 528 of SEQ ID NO: 1, or the amino        acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,        or 80% identity to the amino acid sequence of amino acids 327 to        528 of SEQ ID NO: 1.    -   254. The composition or medical preparation of any one of        embodiments 250 to 253, wherein    -   (i) the RNA encoding a SARS-CoV-2 S protein, an immunogenic        variant thereof, or an immunogenic fragment of the SARS-CoV-2 S        protein or the immunogenic variant thereof comprises the        nucleotide sequence of nucleotides 111 to 986 of SEQ ID NO: 30,        a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%,        90%, 85%, or 80% identity to the nucleotide sequence of        nucleotides 111 to 986 of SEQ ID NO: 30, or a fragment of the        nucleotide sequence of nucleotides 111 to 986 of SEQ ID NO: 30,        or the nucleotide sequence having at least 99%, 98%, 97%, 96%,        95%, 90%, 85%, or 80% identity to the nucleotide sequence of        nucleotides 111 to 986 of SEQ ID NO: 30; and/or    -   (ii) a SARS-CoV-2 S protein, an immunogenic variant thereof, or        an immunogenic fragment of the SARS-CoV-2 S protein or the        immunogenic variant thereof comprises the amino acid sequence of        amino acids 20 to 311 of SEQ ID NO: 29, an amino acid sequence        having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%        identity to the amino acid sequence of amino acids 20 to 311 of        SEQ ID NO: 29, or an immunogenic fragment of the amino acid        sequence of amino acids 20 to 311 of SEQ ID NO: 29, or the amino        acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,        or 80% identity to the amino acid sequence of amino acids 20 to        311 of SEQ ID NO: 29.    -   255. The composition or medical preparation of any one of        embodiments 250 to 254, wherein    -   (i) the RNA encoding a SARS-CoV-2 S protein, an immunogenic        variant thereof, or an immunogenic fragment of the SARS-CoV-2 S        protein or the immunogenic variant thereof comprises the        nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8        or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%,        95%, 90%, 85%, or 80% identity to the nucleotide sequence of        nucleotides 49 to 3819 of SEQ ID NO: 2, 8 or 9, or a fragment of        the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO:        2, 8 or 9, or the nucleotide sequence having at least 99%, 98%,        97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide        sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8 or 9;        and/or    -   (ii) a SARS-CoV-2 S protein, an immunogenic variant thereof, or        an immunogenic fragment of the SARS-CoV-2 S protein or the        immunogenic variant thereof comprises the amino acid sequence of        amino acids 17 to 1273 of SEQ ID NO: 1 or 7, an amino acid        sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or        80% identity to the amino acid sequence of amino acids 17 to        1273 of SEQ ID NO: 1 or 7, or an immunogenic fragment of the        amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or        7, or the amino acid sequence having at least 99%, 98%, 97%,        96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence        of amino acids 17 to 1273 of SEQ ID NO: 1 or 7.    -   256. The composition or medical preparation of any one of        embodiments 250 to 255, wherein the amino acid sequence        comprising a SARS-CoV-2 S protein, an immunogenic variant        thereof, or an immunogenic fragment of the SARS-CoV-2 S protein        or the immunogenic variant thereof comprises a secretory signal        peptide.    -   257. The composition or medical preparation of embodiment 256,        wherein the secretory signal peptide is fused, preferably        N-terminally, to a SARS-CoV-2 S protein, an immunogenic variant        thereof, or an immunogenic fragment of the SARS-CoV-2 S protein        or the immunogenic variant thereof.    -   258. The composition or medical preparation of embodiment 256 or        257, wherein    -   (i) the RNA encoding the secretory signal peptide comprises the        nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8 or        9, a nucleotide sequence having at least 99%, 98%, 97%, 96%,        95%, 90%, 85%, or 80% identity to the nucleotide sequence of        nucleotides 1 to 48 of SEQ ID NO: 2, 8 or 9, or a fragment of        the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2,        8 or 9, or the nucleotide sequence having at least 99%, 98%,        97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide        sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8 or 9; and/or    -   (ii) the secretory signal peptide comprises the amino acid        sequence of amino acids 1 to 16 of SEQ ID NO: 1, an amino acid        sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or        80% identity to the amino acid sequence of amino acids 1 to 16        of SEQ ID NO: 1, or a functional fragment of the amino acid        sequence of amino acids 1 to 16 of SEQ ID NO: 1, or the amino        acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,        or 80% identity to the amino acid sequence of amino acids 1 to        16 of SEQ ID NO: 1.    -   259. The composition or medical preparation of any one of        embodiments 250 to 258, wherein    -   (i) the RNA encoding a SARS-CoV-2 S protein, an immunogenic        variant thereof, or an immunogenic fragment of the SARS-CoV-2 S        protein or the immunogenic variant thereof comprises the        nucleotide sequence of SEQ ID NO: 6, a nucleotide sequence        having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%        identity to the nucleotide sequence of SEQ ID NO: 6, or a        fragment of the nucleotide sequence of SEQ ID NO: 6, or the        nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%,        90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID        NO: 6; and/or    -   (ii) a SARS-CoV-2 S protein, an immunogenic variant thereof, or        an immunogenic fragment of the SARS-CoV-2 S protein or the        immunogenic variant thereof comprises the amino acid sequence of        SEQ ID NO: 5, an amino acid sequence having at least 99%, 98%,        97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid        sequence of SEQ ID NO: 5, or an immunogenic fragment of the        amino acid sequence of SEQ ID NO: 5, or the amino acid sequence        having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%        identity to the amino acid sequence of SEQ ID NO: 5.    -   260. The composition or medical preparation of any one of        embodiments 250 to 259, wherein    -   (i) the RNA encoding a SARS-CoV-2 S protein, an immunogenic        variant thereof, or an immunogenic fragment of the SARS-CoV-2 S        protein or the immunogenic variant thereof comprises the        nucleotide sequence of nucleotides 54 to 986 of SEQ ID NO: 30, a        nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%,        90%, 85%, or 80% identity to the nucleotide sequence of        nucleotides 54 to 986 of SEQ ID NO: 30, or a fragment of the        nucleotide sequence of nucleotides 54 to 986 of SEQ ID NO: 30,        or the nucleotide sequence having at least 99%, 98%, 97%, 96%,        95%, 90%, 85%, or 80% identity to the nucleotide sequence of        nucleotides 54 to 986 of SEQ ID NO: 30; and/or    -   (ii) a SARS-CoV-2 S protein, an immunogenic variant thereof, or        an immunogenic fragment of the SARS-CoV-2 S protein or the        immunogenic variant thereof comprises the amino acid sequence of        amino acids 1 to 311 of SEQ ID NO: 29, an amino acid sequence        having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%        identity to the amino acid sequence of amino acids 1 to 311 of        SEQ ID NO: 29, or an immunogenic fragment of the amino acid        sequence of amino acids 1 to 311 of SEQ ID NO: 29, or the amino        acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,        or 80% identity to the amino acid sequence of amino acids 1 to        311 of SEQ ID NO: 29.    -   261. The composition or medical preparation of any one of        embodiments 250 to 258, wherein the RNA comprises a modified        nucleoside in place of uridine, in particular wherein the        modified nucleoside is selected from pseudouridine (ψ),        N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U), in        particular wherein the modified nucleoside is        N1-methyl-pseudouridine (m1ψ).    -   262. The composition or medical preparation of any one of        embodiments 250 to 261, wherein the RNA comprises a 5′ cap.    -   263. The composition or medical preparation of any one of        embodiments 250 to 262, wherein the RNA encoding an amino acid        sequence comprising a SARS-CoV-2 S protein, an immunogenic        variant thereof, or an immunogenic fragment of the SARS-CoV-2 S        protein or the immunogenic variant thereof comprises a 5′ UTR        comprising the nucleotide sequence of SEQ ID NO: 12, or a        nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%,        90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID        NO: 12.    -   264. The composition or medical preparation of any one of        embodiments 250 to 263, wherein the RNA encoding an amino acid        sequence comprising a SARS-CoV-2 S protein, an immunogenic        variant thereof, or an immunogenic fragment of the SARS-CoV-2 S        protein or the immunogenic variant thereof comprises a 3′ UTR        comprising the nucleotide sequence of SEQ ID NO: 13, or a        nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%,        90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID        NO: 13.    -   265. The composition or medical preparation of any one of        embodiments 250 to 264, wherein the RNA encoding an amino acid        sequence comprising a SARS-CoV-2 S protein, an immunogenic        variant thereof, or an immunogenic fragment of the SARS-CoV-2 S        protein or the immunogenic variant thereof comprises a poly-A        sequence.    -   266. The composition or medical preparation of embodiment 265,        wherein the poly-A sequence comprises at least 100 nucleotides.    -   267. The composition or medical preparation of embodiment 265 or        266, wherein the poly-A sequence comprises or consists of the        nucleotide sequence of SEQ ID NO: 14.    -   268. The composition or medical preparation of any one of        embodiments 250 to 267, wherein the RNA is formulated or is to        be formulated as a liquid, a solid, or a combination thereof.    -   269. The composition or medical preparation of any one of        embodiments 250 to 268, wherein the RNA is formulated or is to        be formulated for injection.    -   270. The composition or medical preparation of any one of        embodiments 250 to 269, wherein the RNA is formulated or is to        be formulated for intramuscular administration.    -   271. The composition or medical preparation of any one of        embodiments 250 to 270, wherein the RNA is formulated or is to        be formulated as particles.    -   272. The composition or medical preparation of embodiment 271,        wherein the particles are lipid nanoparticles (LNP) or lipoplex        (LPX) particles.    -   273. The composition or medical preparation of embodiment 272,        wherein the LNP particles comprise        ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate),        2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide,        1,2-Distearoyl-sn-glycero-3-phosphocholine, and cholesterol.    -   274. The composition or medical preparation of embodiment 272,        wherein the RNA lipoplex particles are obtainable by mixing the        RNA with liposomes.    -   275. The composition or medical preparation of any one of        embodiments 250 to 274, wherein the RNA is mRNA or saRNA.    -   276. The composition or medical preparation of any one of        embodiments 250 to 275, which is a pharmaceutical composition.    -   277. The composition or medical preparation of any one of        embodiments 250 to 276, which is a vaccine.    -   278. The composition or medical preparation of embodiment 276 or        277, wherein the pharmaceutical composition further comprises        one or more pharmaceutically acceptable carriers, diluents        and/or excipients.    -   279. The composition or medical preparation of any one of        embodiments 250 to 275, which is a kit.    -   280. The composition or medical preparation of embodiment 279,        wherein the RNA and optionally the particle forming components        are in separate vials.    -   281. The composition or medical preparation of embodiment 279 or        280, further comprising instructions for use of the composition        or medical preparation for inducing an immune response against        coronavirus in a subject.    -   282. The composition or medical preparation of any one of        embodiments 250 to 281 for pharmaceutical use.    -   283. The composition or medical preparation of embodiment 282,        wherein the pharmaceutical use comprises inducing an immune        response against coronavirus in a subject.    -   284. The composition or medical preparation of embodiment 282 or        283, wherein the pharmaceutical use comprises a therapeutic or        prophylactic treatment of a coronavirus infection.    -   285. The composition or medical preparation of any one of        embodiments 250 to 284, which is for administration to a human.    -   286. The composition or medical preparation of any one of        embodiments 282 to 285, wherein the coronavirus is a        betacoronavirus.    -   287. The composition or medical preparation of any one of        embodiments 282 to 286, wherein the coronavirus is a        sarbecovirus.    -   288. The composition or medical preparation of any one of        embodiments 282 to 287, wherein the coronavirus is SARS-CoV-2.    -   289. A method of inducing an immune response against coronavirus        in a subject comprising administering to the subject a        composition comprising RNA encoding an amino acid sequence        comprising a SARS-CoV-2 S protein, an immunogenic variant        thereof, or an immunogenic fragment of the SARS-CoV-2 S protein        or the immunogenic variant thereof.    -   290. The method of embodiment 289, wherein an immunogenic        fragment of the SARS-CoV-2 S protein comprises the S1 subunit of        the SARS-CoV-2 S protein, or the receptor binding domain (RBD)        of the S1 subunit of the SARS-CoV-2 S protein.    -   291. The method of any one of embodiments 289 or 290, wherein        the amino acid sequence comprising a SARS-CoV-2 S protein, an        immunogenic variant thereof, or an immunogenic fragment of the        SARS-CoV-2 S protein or the immunogenic variant thereof is        encoded by a coding sequence which is codon-optimized and/or the        G/C content of which is increased compared to wild type coding        sequence, wherein the codon-optimization and/or the increase in        the G/C content preferably does not change the sequence of the        encoded amino acid sequence.    -   292. The method of any one of embodiments 289 to 291, wherein    -   (i) the RNA encoding a SARS-CoV-2 S protein, an immunogenic        variant thereof, or an immunogenic fragment of the SARS-CoV-2 S        protein or the immunogenic variant thereof comprises the        nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2,        8 or 9, a nucleotide sequence having at least 99%, 98%, 97%,        96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence        of nucleotides 979 to 1584 of SEQ ID NO: 2, 8 or 9, or a        fragment of the nucleotide sequence of nucleotides 979 to 1584        of SEQ ID NO: 2, 8 or 9, or the nucleotide sequence having at        least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the        nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2,        8 or 9; and/or    -   (ii) a SARS-CoV-2 S protein, an immunogenic variant thereof, or        an immunogenic fragment of the SARS-CoV-2 S protein or the        immunogenic variant thereof comprises the amino acid sequence of        amino acids 327 to 528 of SEQ ID NO: 1, an amino acid sequence        having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%        identity to the amino acid sequence of amino acids 327 to 528 of        SEQ ID NO: 1, or an immunogenic fragment of the amino acid        sequence of amino acids 327 to 528 of SEQ ID NO: 1, or the amino        acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,        or 80% identity to the amino acid sequence of amino acids 327 to        528 of SEQ ID NO: 1.    -   293. The method of any one of embodiments 289 to 292, wherein    -   (i) the RNA encoding a SARS-CoV-2 S protein, an immunogenic        variant thereof, or an immunogenic fragment of the SARS-CoV-2 S        protein or the immunogenic variant thereof comprises the        nucleotide sequence of nucleotides 111 to 986 of SEQ ID NO: 30,        a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%,        90%, 85%, or 80% identity to the nucleotide sequence of        nucleotides 111 to 986 of SEQ ID NO: 30, or a fragment of the        nucleotide sequence of nucleotides 111 to 986 of SEQ ID NO: 30,        or the nucleotide sequence having at least 99%, 98%, 97%, 96%,        95%, 90%, 85%, or 80% identity to the nucleotide sequence of        nucleotides 111 to 986 of SEQ ID NO: 30; and/or    -   (ii) a SARS-CoV-2 S protein, an immunogenic variant thereof, or        an immunogenic fragment of the SARS-CoV-2 S protein or the        immunogenic variant thereof comprises the amino acid sequence of        amino acids 20 to 311 of SEQ ID NO: 29, an amino acid sequence        having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%        identity to the amino acid sequence of amino acids 20 to 311 of        SEQ ID NO: 29, or an immunogenic fragment of the amino acid        sequence of amino acids 20 to 311 of SEQ ID NO: 29, or the amino        acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,        or 80% identity to the amino acid sequence of amino acids 20 to        311 of SEQ ID NO: 29.    -   294. The method of any one of embodiments 289 to 293, wherein    -   (i) the RNA encoding a SARS-CoV-2 S protein, an immunogenic        variant thereof, or an immunogenic fragment of the SARS-CoV-2 S        protein or the immunogenic variant thereof comprises the        nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8        or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%,        95%, 90%, 85%, or 80% identity to the nucleotide sequence of        nucleotides 49 to 3819 of SEQ ID NO: 2, 8 or 9, or a fragment of        the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO:        2, 8 or 9, or the nucleotide sequence having at least 99%, 98%,        97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide        sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8 or 9;        and/or    -   (ii) a SARS-CoV-2 S protein, an immunogenic variant thereof, or        an immunogenic fragment of the SARS-CoV-2 S protein or the        immunogenic variant thereof comprises the amino acid sequence of        amino acids 17 to 1273 of SEQ ID NO: 1 or 7, an amino acid        sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or        80% identity to the amino acid sequence of amino acids 17 to        1273 of SEQ ID NO: 1 or 7, or an immunogenic fragment of the        amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or        7, or the amino acid sequence having at least 99%, 98%, 97%,        96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence        of amino acids 17 to 1273 of SEQ ID NO: 1 or 7.    -   295. The method of any one of embodiments 289 to 294, wherein        the amino acid sequence comprising a SARS-CoV-2 S protein, an        immunogenic variant thereof, or an immunogenic fragment of the        SARS-CoV-2 S protein or the immunogenic variant thereof        comprises a secretory signal peptide.    -   296. The method of embodiment 295, wherein the secretory signal        peptide is fused, preferably N-terminally, to a SARS-CoV-2 S        protein, an immunogenic variant thereof, or an immunogenic        fragment of the SARS-CoV-2 S protein or the immunogenic variant        thereof.    -   297. The method of embodiment 295 or 297, wherein    -   (i) the RNA encoding the secretory signal peptide comprises the        nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8 or        9, a nucleotide sequence having at least 99%, 98%, 97%, 96%,        95%, 90%, 85%, or 80% identity to the nucleotide sequence of        nucleotides 1 to 48 of SEQ ID NO: 2, 8 or 9, or a fragment of        the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2,        8 or 9, or the nucleotide sequence having at least 99%, 98%,        97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide        sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8 or 9; and/or    -   (ii) the secretory signal peptide comprises the amino acid        sequence of amino acids 1 to 16 of SEQ ID NO: 1, an amino acid        sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or        80% identity to the amino acid sequence of amino acids 1 to 16        of SEQ ID NO: 1, or a functional fragment of the amino acid        sequence of amino acids 1 to 16 of SEQ ID NO: 1, or the amino        acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,        or 80% identity to the amino acid sequence of amino acids 1 to        16 of SEQ ID NO: 1.    -   298. The method of any one of embodiments 289 to 297, wherein    -   (i) the RNA encoding a SARS-CoV-2 S protein, an immunogenic        variant thereof, or an immunogenic fragment of the SARS-CoV-2 S        protein or the immunogenic variant thereof comprises the        nucleotide sequence of SEQ ID NO: 6, a nucleotide sequence        having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%        identity to the nucleotide sequence of SEQ ID NO: 6, or a        fragment of the nucleotide sequence of SEQ ID NO: 6, or the        nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%,        90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID        NO: 6; and/or    -   (ii) a SARS-CoV-2 S protein, an immunogenic variant thereof, or        an immunogenic fragment of the SARS-CoV-2 S protein or the        immunogenic variant thereof comprises the amino acid sequence of        SEQ ID NO: 5, an amino acid sequence having at least 99%, 98%,        97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid        sequence of SEQ ID NO: 5, or an immunogenic fragment of the        amino acid sequence of SEQ ID NO: 5, or the amino acid sequence        having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%        identity to the amino acid sequence of SEQ ID NO: 5.    -   299. The method of any one of embodiments 289 to 298, wherein    -   (i) the RNA encoding a SARS-CoV-2 S protein, an immunogenic        variant thereof, or an immunogenic fragment of the SARS-CoV-2 S        protein or the immunogenic variant thereof comprises the        nucleotide sequence of nucleotides 54 to 986 of SEQ ID NO: 30, a        nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%,        90%, 85%, or 80% identity to the nucleotide sequence of        nucleotides 54 to 986 of SEQ ID NO: 30, or a fragment of the        nucleotide sequence of nucleotides 54 to 986 of SEQ ID NO: 30,        or the nucleotide sequence having at least 99%, 98%, 97%, 96%,        95%, 90%, 85%, or 80% identity to the nucleotide sequence of        nucleotides 54 to 986 of SEQ ID NO: 30; and/or    -   (ii) a SARS-CoV-2 S protein, an immunogenic variant thereof, or        an immunogenic fragment of the SARS-CoV-2 S protein or the        immunogenic variant thereof comprises the amino acid sequence of        amino acids 1 to 311 of SEQ ID NO: 29, an amino acid sequence        having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%        identity to the amino acid sequence of amino acids 1 to 311 of        SEQ ID NO: 29, or an immunogenic fragment of the amino acid        sequence of amino acids 1 to 311 of SEQ ID NO: 29, or the amino        acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,        or 80% identity to the amino acid sequence of amino acids 1 to        311 of SEQ ID NO: 29.    -   300. The method of any one of embodiments 289 to 298, wherein        the RNA comprises a modified nucleoside in place of uridine, in        particular wherein the modified nucleoside is selected from        pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ)), and        5-methyl-uridine (m5U), in particular wherein the modified        nucleoside is N1-methyl-pseudouridine (m1ψ).    -   301. The method of any one of embodiments 289 to 300, wherein        the RNA comprises a cap.    -   302. The method of any one of embodiments 289 to 301, wherein        the RNA encoding an amino acid sequence comprising a SARS-CoV-2        S protein, an immunogenic variant thereof, or an immunogenic        fragment of the SARS-CoV-2 S protein or the immunogenic variant        thereof comprises a 5′ UTR comprising the nucleotide sequence of        SEQ ID NO: 12, or a nucleotide sequence having at least 99%,        98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide        sequence of SEQ ID NO: 12.    -   303. The method of any one of embodiments 289 to 302, wherein        the RNA encoding an amino acid sequence comprising a SARS-CoV-2        S protein, an immunogenic variant thereof, or an immunogenic        fragment of the SARS-CoV-2 S protein or the immunogenic variant        thereof comprises a 3′ UTR comprising the nucleotide sequence of        SEQ ID NO: 13, or a nucleotide sequence having at least 99%,        98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide        sequence of SEQ ID NO: 13.    -   304. The method of any one of embodiments 289 to 303, wherein        the RNA encoding an amino acid sequence comprising a SARS-CoV-2        S protein, an immunogenic variant thereof, or an immunogenic        fragment of the SARS-CoV-2 S protein or the immunogenic variant        thereof comprises a poly-A sequence.    -   305. The method of embodiment 304, wherein the poly-A sequence        comprises at least 100 nucleotides.    -   306. The method of embodiment 304 or 305, wherein the poly-A        sequence comprises or consists of the nucleotide sequence of SEQ        ID NO: 14.    -   307. The method of any one of embodiments 289 to 306, wherein        the RNA is formulated as a liquid, a solid, or a combination        thereof.    -   308. The method of any one of embodiments 289 to 307, wherein        the RNA is administered by injection.    -   309. The method of any one of embodiments 289 to 308, wherein        the RNA is administered by intramuscular administration.    -   310. The method of any one of embodiments 289 to 309, wherein        the RNA is formulated as particles.    -   311. The method of embodiment 310, wherein the particles are        lipid nanoparticles (LNP) or lipoplex (LPX) particles.    -   312. The method of embodiment 311, wherein the LNP particles        comprise        ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate),        2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide,        1,2-Distearoyl-sn-glycero-3-phosphocholine, and cholesterol.    -   313. The method of any one of embodiment 311, wherein the RNA        lipoplex particles are obtainable by mixing the RNA with        liposomes.    -   314. The method of any one of embodiments 289 to 313, wherein        the RNA is mRNA or saRNA.    -   315. The method of any one of embodiments 289 to 314, which is a        method for vaccination against coronavirus.    -   316. The method of any one of embodiments 289 to 315, which is a        method for therapeutic or prophylactic treatment of a        coronavirus infection.    -   317. The method of any one of embodiments 289 to 316, wherein        the subject is a human.    -   318. The method of any one of embodiments 289 to 317, wherein        the coronavirus is a betacoronavirus.    -   319. The method of any one of embodiments 289 to 318, wherein        the coronavirus is a sarbecovirus.    -   320. The method of any one of embodiments 289 to 319, wherein        the coronavirus is SARS-CoV-2.    -   321. The method of any one of embodiments 289 to 320, wherein        the composition is a composition of any one of embodiments 1 to        39.    -   322. A composition or medical preparation of any one of        embodiments 250 to 288 for use in a method of any one of        embodiments 289 to 320.    -   323. An immunogenic composition comprising: a lipid nanoparticle        (LNP) comprising an RNA, wherein the RNA encodes the polypeptide        of SEQ ID NO: 49 and comprises the nucleotide sequence of SEQ ID        NO: 50 or a nucleotide sequence that is at least 80% (e.g., at        85%, at least 90%, at least 91%, at least 92%, at least 93%, at        least 94%, at least 95%, at least 97%, at least 98%, or 99% or        higher) identical to SEQ ID NO: 50, and wherein the RNA        comprises:        -   (a) modified uridines;        -   (b) a 5′ cap; and        -   wherein the LNP comprises            ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate),            2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide,            1,2-Distearoyl-sn-glycero-3-phosphocholine, and cholesterol.    -   324. The immunogenic composition of embodiment 323, wherein the        nucleotide sequence includes modified uridines in place of all        uridines.    -   325. The immunogenic of embodiment 323 or 324, wherein the        modified uridines are each N1-methyl-pseudouridine.    -   326. The immunogenic composition of any one of embodiments 323        to 325, wherein the RNA further comprises at least one, at least        two, or all of the following features:        -   a 5′ untranslated region (UTR) comprising SEQ ID NO: 12;        -   a 3′ untranslated region (UTR) comprising SEQ ID NO: 13; and        -   a poly-A sequence of at least 100 A nucleotides.    -   327. The immunogenic composition of embodiment 326, wherein the        poly-A sequence comprises 30 adenine nucleotides followed by 70        adenine nucleotides, wherein the 30 adenine nucleotides and 70        adenine nucleotides are separated by a linker sequence.    -   328. The immunogenic composition of embodiment 326, wherein the        poly-A sequence comprises SEQ ID NO: 14.    -   329. The immunogenic composition of any one of embodiments 323        to 328, wherein the RNA comprises SEQ ID NO: 51.    -   330. An immunogenic composition comprising a lipid nanoparticle        (LNP) comprising an RNA, wherein the RNA encodes the polypeptide        of SEQ ID NO: 55 and comprises the nucleotide sequence of SEQ ID        NO: 56 or a nucleotide sequence that is at least 80% (e.g., at        85%, at least 90%, at least 91%, at least 92%, at least 93%, at        least 94%, at least 95%, at least 97%, at least 98%, or 99% or        higher) identical to SEQ ID NO: 56, and wherein the RNA        comprises:        -   (a) modified uridines;        -   (b) a 5′ cap; and        -   wherein the LNP comprises            ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate),            2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide,            1,2-Distearoyl-sn-glycero-3-phosphocholine, and cholesterol.    -   331. The immunogenic composition of embodiment 330, wherein the        nucleotide sequence includes modified uridines in place of all        uridines.    -   332. The immunogenic of embodiment 330 or 331, wherein the        modified uridines are each N1-methyl-pseudouridine.    -   333. The immunogenic composition of any one of embodiments 330        to 332, wherein the RNA further comprises at least one, at least        two, or all of the following features:        -   a 5′ untranslated region (UTR) comprising SEQ ID NO: 12;        -   a 3′ untranslated region (UTR) comprising SEQ ID NO: 13; and            a poly-A sequence of at least 100 A nucleotides.    -   334. The immunogenic composition of embodiment 333, wherein the        poly-A sequence comprises 30 adenine nucleotides followed by 70        adenine nucleotides, wherein the 30 adenine nucleotides and 70        adenine nucleotides are separated by a linker sequence.    -   335. The immunogenic composition of embodiment 334, wherein the        poly-A sequence comprises SEQ ID NO: 14.    -   336. The immunogenic composition of any one of embodiments 330        to 335, wherein the RNA comprises SEQ ID NO: 57.    -   337. An immunogenic composition comprising a lipid nanoparticle        (LNP) comprising an RNA, wherein the RNA encodes the polypeptide        of SEQ ID NO: 58 and comprises the nucleotide sequence of SEQ ID        NO: 59 or a nucleotide sequence that is at least 80% (e.g., at        85%, at least 90%, at least 91%, at least 92%, at least 93%, at        least 94%, at least 95%, at least 97%, at least 98%, or 99% or        higher) identical to SEQ ID NO: 59, and wherein the RNA        comprises:        -   (a) modified uridines;        -   (b) a 5′ cap; and        -   wherein the LNP comprises            ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate),            2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide,            1,2-Distearoyl-sn-glycero-3-phosphocholine, and cholesterol.    -   338. The immunogenic composition of embodiment 337, wherein the        nucleotide sequence includes modified uridines in place of all        uridines.    -   339. The immunogenic of embodiment 337 or 338, wherein the        modified uridines are each N1-methyl-pseudouridine.    -   340. The immunogenic composition of any one of embodiments 337        to 339, wherein the RNA further comprises at least one, at least        two, or all of the following features:        -   a 5′ untranslated region (UTR) comprising SEQ ID NO: 12;        -   a 3′ untranslated region (UTR) comprising SEQ ID NO: 13; and        -   a poly-A sequence of at least 100 A nucleotides.    -   341. The immunogenic composition of embodiment 340, wherein the        poly-A sequence comprises 30 adenine nucleotides followed by 70        adenine nucleotides, wherein the 30 adenine nucleotides and 70        adenine nucleotides are separated by a linker sequence.    -   342. The immunogenic composition of embodiment 341, wherein the        poly-A sequence comprises SEQ ID NO: 14.    -   343. The immunogenic composition of any one of embodiments 337        to 342, wherein the RNA comprises SEQ ID NO: 60.    -   344. An immunogenic composition comprising a lipid nanoparticle        (LNP) comprising an RNA, wherein the RNA encodes the polypeptide        of SEQ ID NO: 61 and comprises the nucleotide sequence of SEQ ID        NO: 62a or a nucleotide sequence that is at least 80% (e.g., at        85%, at least 90%, at least 91%, at least 92%, at least 93%, at        least 94%, at least 95%, at least 97%, at least 98%, or 99% or        higher) identical to SEQ ID NO: 62a, and wherein the RNA        comprises:        -   (a) modified uridines;        -   (b) a 5′ cap; and        -   wherein the LNP comprises            ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate),            2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide,            1,2-Distearoyl-sn-glycero-3-phosphocholine, and cholesterol.    -   345. The immunogenic composition of embodiment 344, wherein the        nucleotide sequence includes modified uridines in place of all        uridines.    -   346. The immunogenic of embodiment 344 or 345, wherein the        modified uridines are each N1-methyl-pseudouridine.    -   347. The immunogenic composition of any one of embodiments 344        to 346, wherein the RNA further comprises at least one, at least        two, or all of the following features:        -   a 5′ untranslated region (UTR) comprising SEQ ID NO: 12;        -   a 3′ untranslated region (UTR) comprising SEQ ID NO: 13; and            a poly-A sequence of at least 100 A nucleotides.    -   348. The immunogenic composition of embodiment 347, wherein the        poly-A sequence comprises 30 adenine nucleotides followed by 70        adenine nucleotides, wherein the 30 adenine nucleotides and 70        adenine nucleotides are separated by a linker sequence.    -   349. The immunogenic composition of embodiment 348, wherein the        poly-A sequence comprises SEQ ID NO: 14.    -   350. The immunogenic composition of any one of embodiments 344        to 349, wherein the RNA comprises SEQ ID NO: 63a.    -   351. An immunogenic composition comprising a first RNA and a        second RNA, wherein:        -   the first RNA encodes the polypeptide of SEQ ID NO: 7 and            comprises the nucleotide sequence of SEQ ID NO: 9 or a            nucleotide sequence that is at least 80% (e.g., at 85%, at            least 90%, at least 91%, at least 92%, at least 93%, at            least 94%, at least 95%, at least 97%, at least 98%, or 99%            or higher) identical to SEQ ID NO: 9, and        -   the second RNA encodes the polypeptide of SEQ ID NO: 49 and            comprises the nucleotide sequence of SEQ ID NO: 50 or a            nucleotide sequence that is at least 80% (e.g., at 85%, at            least 90%, at least 91%, at least 92%, at least 93%, at            least 94%, at least 95%, at least 97%, at least 98%, or 99%            or higher) identical to SEQ ID NO: 50, and        -   wherein each of the first RNA and the second RNA comprise:        -   (a) modified uridines; and        -   (b) a 5′ cap, and        -   wherein the first RNA and the second RNA are formulated in            lipid nanoparticles (LNPs), wherein the LNPs comprise            ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate),            2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide,            1,2-Distearoyl-sn-glycero-3-phosphocholine, and cholesterol.    -   352. The immunogenic composition of embodiment 351, wherein the        first RNA and the second RNA are formulated in the same lipid        nanoparticles.    -   353. The immunogenic composition of embodiment 351, wherein the        first RNA and the second RNA are formulated in separate lipid        nanoparticles.    -   354. The immunogenic composition of any one of embodiments 351        to 353, wherein each of the first RNA and the second RNA include        modified uridines in place of all uridines.    -   355. The immunogenic of any one of embodiments 351 to 354,        wherein the modified uridines are each N1-methyl-pseudouridine.    -   356. The immunogenic composition of any one of embodiments 351        to 355, wherein the first RNA and the second RNA each        independently comprise at least one, at least two, or all of the        following features:        -   a 5′ untranslated region (UTR) comprising SEQ ID NO: 12;        -   a 3′ untranslated region (UTR) comprising SEQ ID NO: 13; and            a poly-A sequence of at least 100 A nucleotides.    -   357. The immunogenic composition of embodiment 357, wherein the        poly-A sequence comprises 30 adenine nucleotides followed by 70        adenine nucleotides, wherein the 30 adenine nucleotides and 70        adenine nucleotides are separated by a linker sequence.    -   358. The immunogenic composition of embodiment 357, wherein the        poly-A sequence comprises SEQ ID NO: 14.    -   359. The immunogenic composition of any one of embodiments 351        to 358, wherein the first RNA comprises SEQ ID NO: 20 and the        second RNA comprises SEQ ID NO: 51.    -   360. An immunogenic composition comprising a first RNA and a        second RNA, wherein:        -   the first RNA encodes the polypeptide of SEQ ID NO: 7 and            comprises the nucleotide sequence of SEQ ID NO: 9 or a            nucleotide sequence that is at least 80% (e.g., at 85%, at            least 90%, at least 91%, at least 92%, at least 93%, at            least 94%, at least 95%, at least 97%, at least 98%, or 99%            or higher) identical to SEQ ID NO: 9, and        -   the second RNA encodes the polypeptide of SEQ ID NO: 55, 58,            or 61 and comprises the nucleotide sequence of SEQ ID NO:            56, 59, or 62a, or a nucleotide sequence that is at least            80% (e.g., at 85%, at least 90%, at least 91%, at least 92%,            at least 93%, at least 94%, at least 95%, at least 97%, at            least 98%, or 99% or higher) identical to SEQ ID NO: 56, 59,            or 62a, and        -   wherein each of the first RNA and the second RNA comprise:        -   (a) modified uridines; and        -   (b) a 5′ cap, and        -   wherein the first RNA and the second RNA are formulated in            lipid nanoparticles (LNPs), wherein the LNPs comprise            ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate),            2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide,            1,2-Distearoyl-sn-glycero-3-phosphocholine, and cholesterol.    -   361. The immunogenic composition of embodiment 360, wherein the        first RNA and the second RNA are formulated in separate lipid        nanoparticles.    -   362. The immunogenic composition of embodiment 360, wherein the        first RNA and the second RNA are formulated in the same lipid        nanoparticles.    -   363. The immunogenic composition of any one of embodiments 360        to 362, wherein the first RNA and the second RNA each include        modified uridines in place of all uridines.    -   364. The immunogenic composition of any one of embodiments 360        to 363, wherein the modified uridines are each        N1-methyl-pseudouridine.    -   365. The immunogenic composition of any one of embodiments 360        to 364, wherein the first RNA and the second RNA each        independently further comprise at least one, at least two, or        all of the following features:        -   a 5′ untranslated region (UTR) comprising SEQ ID NO: 12;        -   a 3′ untranslated region (UTR) comprising SEQ ID NO: 13; and        -   a poly-A sequence of at least 100 A nucleotides.    -   366. The immunogenic composition of any one of embodiments 360        to 365, wherein the poly-A sequence comprises 30 adenine        nucleotides followed by 70 adenine nucleotides, wherein the 30        adenine nucleotides and 70 adenine nucleotides are separated by        a linker sequence.    -   367. The immunogenic composition of embodiment 366, wherein the        poly-A sequence comprises SEQ ID NO: 14.    -   368. The immunogenic composition of any one of embodiments 360        to 367, wherein the first RNA comprises SEQ ID NO: 9 and the        second RNA comprises SEQ ID NO: 56.    -   369. The immunogenic composition of any one of embodiments 360        to 368, wherein the first RNA comprises SEQ ID NO: 9 and the        second RNA comprises SEQ ID NO: 59.    -   370. The immunogenic composition of any one of embodiments 360        to 368, wherein the first RNA comprises SEQ ID NO: 9 and the        second RNA comprises SEQ ID NO: 62a.    -   371. The immunogenic composition of any one of embodiments 360        to 368, wherein the first RNA comprises SEQ ID NO: 20 and the        second RNA comprises SEQ ID NO: 57.    -   372. The immunogenic composition of any one of embodiments 360        to 368, wherein the first RNA comprises SEQ ID NO: 20 and the        second RNA comprises SEQ ID NO: 60.    -   373. The immunogenic composition of any one of embodiments 360        to 368, wherein the first RNA comprises SEQ ID NO: 20 and the        second RNA comprises SEQ ID NO: 63a.    -   374. An immunogenic composition comprising a first RNA and a        second RNA, wherein:        -   the first RNA encodes the polypeptide of SEQ ID NO: 58 and            comprises the nucleotide sequence of SEQ ID NO: 59 or a            nucleotide sequence that is at least 80% (e.g., at 85%, at            least 90%, at least 91%, at least 92%, at least 93%, at            least 94%, at least 95%, at least 97%, at least 98%, or 99%            or higher) identical to SEQ ID NO: 59, and        -   the second RNA encodes the polypeptide of SEQ ID NO: 49, 55,            or 61 and comprises the nucleotide sequence of SEQ ID NO:            50, 56, or 62a, or a nucleotide sequence that is at least            80% (e.g., at 85%, at least 90%, at least 91%, at least 92%,            at least 93%, at least 94%, at least 95%, at least 97%, at            least 98%, or 99% or higher) identical to SEQ ID NO: 50, 56,            or 62a, and        -   wherein each of the first RNA and the second RNA comprise:        -   (a) modified uridines; and        -   (b) a 5′ cap, and        -   wherein the first RNA and the second RNA are formulated in            lipid nanoparticles (LNPs), wherein the LNPs comprise            ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate),            2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide,            1,2-Distearoyl-sn-glycero-3-phosphocholine, and cholesterol.    -   375. The immunogenic composition of embodiment 374, wherein the        first RNA and the second RNA are formulated in separate lipid        nanoparticles.    -   376. The immunogenic composition of embodiment 374, wherein the        first RNA and the second RNA are formulated in the same lipid        nanoparticles.    -   377. The immunogenic composition of any one of embodiments 374        to 376, wherein the first RNA and the second RNA each include        modified uridines in place of all uridines.    -   378. The immunogenic of any one of embodiments 374 to 377,        wherein the modified uridines are each N1-methyl-pseudouridine.    -   379. The immunogenic composition of any one of embodiments 374        to 378, wherein the first RNA and the second RNA each        independently further comprise at least one, at least two, or        all of the following features:        -   a 5′ untranslated region (UTR) comprising SEQ ID NO: 12;        -   a 3′ untranslated region (UTR) comprising SEQ ID NO: 13; and            a poly-A sequence of at least 100 A nucleotides.    -   380. The immunogenic composition of embodiment 379, wherein the        poly-A sequence comprises 30 adenine nucleotides followed by 70        adenine nucleotides, wherein the 30 adenine nucleotides and 70        adenine nucleotides are separated by a linker sequence.    -   381. The immunogenic composition of embodiment 379, wherein the        poly-A sequence comprises SEQ ID NO: 14.    -   382. The immunogenic composition of any one of embodiments 374        to 381, wherein the first RNA comprises SEQ ID NO: 59 and the        second RNA comprises SEQ ID NO: 50.    -   383. The immunogenic composition of any one of embodiments 374        to 381, wherein the first RNA comprises SEQ ID NO: 59 and the        second RNA comprises SEQ ID NO: 56.    -   384. The immunogenic composition of any one of embodiments 374        to 381, wherein the first RNA comprises SEQ ID NO: 59 and the        second RNA comprises SEQ ID NO: 62a.    -   385. The immunogenic composition of any one of embodiments 374        to 381, wherein the first RNA comprises SEQ ID NO: 60 and the        second RNA comprises SEQ ID NO: 51.    -   386. The immunogenic composition of any one of embodiments 374        to 381, wherein the first RNA comprises SEQ ID NO: 60 and the        second RNA comprises SEQ ID NO: 57.    -   387. The immunogenic composition of any one of embodiments 374        to 381, wherein the first RNA comprises SEQ ID NO: 60 and the        second RNA comprises SEQ ID NO: 63a.    -   388. An immunogenic composition comprising a first RNA and a        second RNA, wherein:        -   the first RNA encodes the polypeptide of SEQ ID NO: 49 and            comprises the nucleotide sequence of SEQ ID NO: 50 or a            nucleotide sequence that is at least 80% (e.g., at 85%, at            least 90%, at least 91%, at least 92%, at least 93%, at            least 94%, at least 95%, at least 97%, at least 98%, or 99%            or higher) identical to SEQ ID NO: 50, and        -   the second RNA encodes the polypeptide of SEQ ID NO: 55 or            61 and comprises the nucleotide sequence of SEQ ID NO: 56 or            62a, or a nucleotide sequence that is at least 80% (e.g., at            85%, at least 90%, at least 91%, at least 92%, at least 93%,            at least 94%, at least 95%, at least 97%, at least 98%, or            99% or higher) identical to SEQ ID NO: 56 or 62a, and        -   wherein each of the first RNA and the second RNA comprise:        -   (a) modified uridines; and        -   (b) a 5′ cap, and        -   wherein the first RNA and the second RNA are formulated in            lipid nanoparticles (LNPs), wherein the LNPs comprise            ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate),            2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide,            1,2-Distearoyl-sn-glycero-3-phosphocholine, and cholesterol.    -   389. The immunogenic composition of embodiment 388, wherein the        first RNA and the second RNA are formulated in separate lipid        nanoparticles.    -   390. The immunogenic composition of embodiment 388, wherein the        first RNA and the second RNA are formulated in the same lipid        nanoparticles.    -   391. The immunogenic composition of any one of embodiments 388        to 390, wherein the first RNA and the second RNA each include        modified uridines in place of all uridines.    -   392. The immunogenic of any one of embodiments 388 to 391,        wherein the modified uridines are each N1-methyl-pseudouridine.    -   393. The immunogenic composition of any one of embodiments 388        to 392, wherein the first RNA and the second RNA further each        independently further comprise at least one, at least two, or        all of the following features:        -   a 5′ untranslated region (UTR) comprising SEQ ID NO: 12;        -   a 3′ untranslated region (UTR) comprising SEQ ID NO: 13; and        -   a poly-A sequence of at least 100 A nucleotides.    -   394. The immunogenic composition of embodiment 393, wherein the        poly-A sequence comprises 30 adenine nucleotides followed by 70        adenine nucleotides, wherein the 30 adenine nucleotides and 70        adenine nucleotides are separated by a linker sequence.    -   395. The immunogenic composition of embodiment 393, wherein the        poly-A sequence comprises SEQ ID NO: 14.    -   396. The immunogenic composition of any one of embodiments 388        to 395, wherein the first RNA comprises SEQ ID NO: 50 and the        second RNA comprises SEQ ID NO: 56.    -   397. The immunogenic composition of any one of embodiments 388        to 395, wherein the first RNA comprises SEQ ID NO: 50 and the        second RNA comprises SEQ ID NO: 62a.    -   398. The immunogenic composition of any one of embodiments 388        to 395, wherein the first RNA comprises SEQ ID NO: 51 and the        second RNA comprises SEQ ID NO: 57.    -   399. The immunogenic composition of any one of embodiments 388        to 395, wherein the first RNA comprises SEQ ID NO: 51 and the        second RNA comprises SEQ ID NO: 63a.    -   400. An immunogenic composition comprising a first RNA and a        second RNA, wherein:        -   the first RNA encodes the polypeptide of SEQ ID NO: 55 and            comprises the nucleotide sequence of SEQ ID NO: 56 or a            nucleotide sequence that is at least 80% (e.g., at 85%, at            least 90%, at least 91%, at least 92%, at least 93%, at            least 94%, at least 95%, at least 97%, at least 98%, or 99%            or higher) identical to SEQ ID NO: 56, and        -   the second RNA encodes the polypeptide of SEQ ID NO: 61 and            comprises the nucleotide sequence of SEQ ID NO: 62a, or a            nucleotide sequence that is at least 80% (e.g., at 85%, at            least 90%, at least 91%, at least 92%, at least 93%, at            least 94%, at least 95%, at least 97%, at least 98%, or 99%            or higher) identical to SEQ ID NO: 62a, and        -   wherein each of the first RNA and the second RNA comprise:        -   (a) modified uridines; and        -   (b) a 5′ cap, and        -   wherein the first RNA and the second RNA are formulated in            lipid nanoparticles (LNPs), wherein the LNPs comprise            ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate),            2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide,            1,2-Distearoyl-sn-glycero-3-phosphocholine, and cholesterol.    -   401. The immunogenic composition of embodiment 400, wherein the        first RNA and the second RNA are formulated in separate lipid        nanoparticles.    -   402. The immunogenic composition of embodiment 400, wherein the        first RNA and the second RNA are formulated in the same lipid        nanoparticles.    -   403. The immunogenic composition of any one of embodiments 400        to 402, wherein the first RNA and the second RNA each include        modified uridines in place of all uridines.    -   404. The immunogenic of any one of embodiments 400 to 403,        wherein the modified uridines are each N1-methyl-pseudouridine.    -   405. The immunogenic composition of any one of embodiments 400        to 404, wherein the first RNA and the second RNA each        independently further comprise at least one, at least two, or        all of the following features:        -   a 5′ untranslated region (UTR) comprising SEQ ID NO: 12;        -   a 3′ untranslated region (UTR) comprising SEQ ID NO: 13; and            a poly-A sequence of at least 100 A nucleotides.    -   406. The immunogenic composition of embodiment 405, wherein the        poly-A sequence comprises 30 adenine nucleotides followed by 70        adenine nucleotides, wherein the 30 adenine nucleotides and 70        adenine nucleotides are separated by a linker sequence.    -   407. The immunogenic composition of embodiment 405, wherein the        poly-A sequence comprises SEQ ID NO: 14.    -   408. The immunogenic composition of any one of embodiments 400        to 407, wherein the first RNA comprises SEQ ID NO: 57 and the        second RNA comprises SEQ ID NO: 63a.    -   409. The immunogenic composition of any one of embodiments 323        to 408, wherein the 5′-cap is or comprises m₂ ^(7,3′-O)Gppp(m₁        ^(2′-O))ApG.    -   410. The immunogenic composition of any one of embodiments 323        to 409, wherein the LNP comprises about 40 to about 50 mole        percent        ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate),        about 35 to about 45 mole percent cholesterol, about 5 to about        mole percent 1,2-Distearoyl-sn-glycero-3-phosphocholine, and        about 1 to about 10 mole percent 2-[(polyethylene        glycol)-2000]-N,N-ditetradecylacetamide.    -   411. The immunogenic composition of any one of embodiments 323        to 410, wherein the composition comprises a plurality of LNPs,        wherein the average diameter of the plurality of LNPs is about        30 nm to about 200 nm or about 60 nm to about 120 nm (e.g., as        determined by dynamic light scattering measurements).    -   412. A method of eliciting an immune response against SARS-CoV-2        comprising administering the immunogenic composition of any one        of embodiments 74 to 162.    -   413. The method of embodiment 413, wherein the immune response        is elicited against an Omicron variant of SARS-CoV-2.    -   414. The method of embodiment 412, wherein the immune response        is elicited against a Beta variant of SARS-CoV-2.    -   415. The method of embodiment 412, wherein the immune response        is elicited against an Alpha variant of SARS-CoV-2.    -   416. The method of embodiment 412, wherein the immune response        is elicited against a Delta variant of SARS-CoV-2.    -   417. The method of embodiment 412, wherein the immune response        is elicited against a Wuhan strain, an Omicron variant, a Beta        variant, an Alpha variant, and a Delta variant of SARS-CoV-2.    -   418. A method for inducing an immune response in a subject,        wherein the method comprises delivering (e.g., as a polypeptide        or an RNA encoding such a polypeptide) an antigen of a        SARS-CoV-2 virus that is not a BA.1 Omicron variant of        SARS-CoV-2.    -   419. The method of embodiment 418, wherein the subject has        previously been infected with or vaccinated against SARS-CoV-2.    -   420. The method of embodiment 418 or 419, wherein the subject        has previously been delivered (e.g., as a polypeptide or an RNA        encoding such a polypeptide) an antigen of a Wuhan strain of        SARS-CoV-2.    -   421. The method of any one of embodiments 418-420, wherein the        subject has previously been administered RNA encoding a        SARS-CoV-2 S protein of a Wuhan strain.    -   422. The method of any one of embodiments 418-421, wherein the        subject has previously been administered two or more doses of        RNA encoding a SARS-CoV-2 S protein of a Wuhan strain.    -   423. The method of any one of embodiments 418-422, wherein the        method comprises administering RNA encoding an antigen of a        SARS-CoV-2 virus that is not a BA.1 Omicron variant.    -   424. The method of any one of embodiments 418-423, wherein the        method comprises administering RNA encoding a SARS-CoV-2 S        protein from a SARS-CoV-2 variant that is a BA.1 Omicron        variant.    -   425. The method of any one of embodiments 418-424, wherein the        method comprises administering RNA encoding an S protein of an        Omicron variant of SARS-CoV-2, wherein the Omicron variant is        not a BA.1 Omicron variant.    -   426. The method of any one of embodiments 418-425, wherein the        method comprises administering RNA encoding an S protein of a        BA.2 Omicron variant of SARS-CoV-2.    -   427. The method of any one of embodiments 418-425, wherein the        method comprises administering RNA encoding an S protein of a        BA.4 or BA.5 Omicron variant of SARS-CoV-2.    -   428. A method for inducing an immune response in a subject,        wherein the method comprises administering (a) a first RNA        encoding a SARS-CoV-2 S protein of a Wuhan strain, an Alpha        variant, a Beta variant, or a Delta variant, and (b) a second        RNA encoding a SARS-CoV-2 S protein of an Omicron variant that        is not a BA.1 Omicron variant.    -   429. The method of embodiment 428, where the second RNA encodes        an S protein of a BA.2 Omicron variant.    -   430. The method of embodiment 428, where the second RNA encodes        an S protein of a BA.4 or BA.5 Omicron variant.    -   431. A method for inducing an immune response in a subject,        wherein the method comprises administering (a) a first RNA        encoding a SARS-CoV-2 S protein of a Wuhan strain, an Alpha        variant, a Beta variant, a Delta variant, or a BA.1 Omicron        variant and (b) a second RNA encoding a SARS-CoV-2 S protein        that is antigenically distinct from the S protein encoded by the        first RNA.    -   432. The method of embodiment 431, wherein the second RNA        encodes a SARS-CoV-2 S protein of an Omicron variant that is not        a BA.1 Omicron variant.    -   433. The method of embodiment 431, wherein the second RNA        encodes a SARS-CoV-2 S protein of a BA.2 Omicron variant.    -   434. The method of embodiment 431, wherein the second RNA        encodes a SARS-CoV-2 S protein of a BA.4 or BA.5 Omicron        variant.    -   435. The method of any one of embodiments 418-434, wherein the        first RNA and the second RNA are encapsulated in separate LNPs.    -   436. The method of any one of embodiments 418-435, wherein the        first RNA and the second RNA are encapsulated in the same LNP.    -   437. The method of any one of embodiments 418-436, wherein the        first RNA and the second RNA are administered separately, e.g.,        at different injection sites.    -   438. A composition comprising (a) a first RNA encoding a        SARS-CoV-2 S protein of a Wuhan strain, an Alpha variant, a Beta        variant, a Delta variant, or a BA.1 Omicron variant and (b) a        second RNA encoding a SARS-CoV-2 S protein that is antigenically        distinct from the S protein encoded by the first RNA.    -   439. A composition comprising (a) a first RNA encoding a        SARS-CoV-2 S protein of a Wuhan strain, an Alpha variant, a Beta        variant, a Delta variant, or a BA.1 Omicron variant and (b) a        second RNA encoding a SARS-CoV-2 S protein an Omicron variant        that is not a BA.1 Omicron variant.    -   440. The composition of embodiment 439, wherein the second RNA        encodes a SARS-CoV-2 S protein of a BA.2 Omicron variant.    -   441. The composition of embodiment 439, wherein the second RNA        encodes a SARS-CoV-2 S protein of a BA.4 or BA.5 Omicron        variant.    -   442. The composition of embodiment 439, wherein the first RNA        encodes a SARS-CoV-2 S protein of a Wuhan strain and the second        RNA encodes a SARS-CoV-2 S protein of a BA.2 Omicron variant.    -   443. The composition of embodiment 439, wherein the first RNA        encodes a SARS-CoV-2 S protein of a Wuhan strain and the second        RNA encodes a SARS-CoV-2 S protein of a BA.4 or BA.5 Omicron        variant.    -   444. The composition of embodiment 439, wherein the first RNA        encodes a SARS-CoV-2 S protein of a BA.1 Omicron variant and the        second RNA encodes a SARS-CoV-2 S protein of a BA.2 Omicron        variant.    -   445. The composition of embodiment 439, wherein the first RNA        encodes a SARS-CoV-2 S protein of a BA.1 Omicron variant and the        second RNA encodes a SARS-CoV-2 S protein of a BA.4 or BA.5        Omicron variant.    -   446. The composition of any one of embodiments 438-445, wherein        the first RNA and the second RNA are administered in        encapsulated in the same LNP.    -   447. The composition of any one of embodiments 438-446, wherein        the first RNA and the second RNA are encapsulated in separate        LNPs.    -   448. The method of any one of embodiments 214 to 249, 289 to        321, or 412 to 437, wherein the composition is a composition of        any one of embodiments 1 to 213, 250 to 288, 322 to 411, or 438        to 448.    -   448. A composition or medical preparation of any one of        embodiments 1 to 213, 250 to 288, 322 to 411, or 438 to 448 for        use in a method of any one of embodiments 214 to 249, 289 to        321, or 412 to 437.    -   449. The composition or medical preparation of any one of        embodiments 1 to 213, 250 to 288, 322 to 411, or 438 to 448,        which is a pharmaceutical composition.    -   450. The composition or medical preparation of any one of        embodiments 1 to 213, 250 to 288, 322 to 411, or 438 to 448,        which is a vaccine.    -   451. The composition or medical preparation of embodiment 449 or        450, wherein the pharmaceutical composition or vaccine further        comprises one or more pharmaceutically acceptable carriers,        diluents and/or excipients.    -   452. The composition or medical preparation of any one of        embodiment 1 to 213, 250 to 288, 322 to 411, or 438 to 448,        which is a kit.    -   453. The composition or medical preparation of embodiment 452,        wherein the RNA and optionally the particle forming components        are in separate vials.    -   454. The composition or medical preparation of embodiment 452 or        453, further comprising instructions for use of the composition        or medical preparation for inducing an immune response against        coronavirus in a subject.    -   455. The composition or medical preparation of any one of        embodiments 1 to 213, 250 to 288, 322 to 411, or 438 to 448 for        pharmaceutical use.    -   456. The composition or medical preparation of embodiment 455,        wherein the pharmaceutical use comprises inducing an immune        response against coronavirus in a subject.    -   457. The composition or medical preparation of embodiment 455 or        456, wherein the pharmaceutical use comprises a therapeutic or        prophylactic treatment of a coronavirus infection.    -   458. The composition or medical preparation of any one of        embodiments 1 to 213, 250 to 288, 322 to 411, or 438 to 448 for        use in the manufacture of a medicament.    -   459. The composition or medical preparation of embodiment 458,        wherein the medicament is for inducing an immune response        against coronavirus in a subject.    -   460. The composition or medical preparation of embodiment 458 or        459, wherein the medicament is for therapeutic or prophylactic        treatment of a coronavirus infection.    -   461. The composition or medical preparation of any one of        embodiments 1 to 213, 250 to 288, 322 to 411, or 438 to 448,        which is for administration to a human.    -   462. The composition or medical preparation of any one of        embodiments 454, 456, 457, 459, or 460, wherein the coronavirus        is a betacoronavirus.    -   463. The composition or medical preparation of embodiment 462,        wherein the coronavirus is a sarbecovirus.    -   464. The composition or medical preparation of embodiment 463,        wherein the coronavirus is SARS-CoV-2.

Citation of documents and studies referenced herein is not intended asan admission that any of the foregoing is pertinent prior art. Allstatements as to the contents of these documents are based on theinformation available to the applicants and do not constitute anyadmission as to the correctness of the contents of these documents.

The following description is presented to enable a person of ordinaryskill in the art to make and use the various embodiments. Descriptionsof specific devices, techniques, and applications are provided only asexamples. Various modifications to the examples described herein will bereadily apparent to those of ordinary skill in the art, and the generalprinciples defined herein may be applied to other examples andapplications without departing from the spirit and scope of the variousembodiments. Thus, the various embodiments are not intended to belimited to the examples described herein and shown, but are to beaccorded the scope consistent with the claims.

EXAMPLES Example 1: Immunogenicity Study of BNT162b3 Variants BNT162b3cand BNT162b3d

To get an idea about the potential potency of transmembrane-anchoredRBD-based vaccine antigens (Schematic in FIG. 6 ; BNT162b3c (1) andBNT162b3d (2)), BALB/c mice were immunized IM once with 4 μg LNP-C12formulated mRNA or with buffer as control. The non-clinical LNP-C12formulated mRNAs were used as surrogate for the BNT162b3 variantsBNT162b3c and BNT162b3d. The immunogenicity of the RNA vaccine wasinvestigated by focusing on the antibody immune response.

ELISA data 6, 14 and 21 d after the first immunization show an early,dose-dependent immune activation against the S1 protein and the receptorbinding domain (FIG. 7 ). Sera obtained 6, 14 and 21 d afterimmunization show high SARS-CoV-2 pseudovirus neutralization,correlating with the increase of IgG antibody titers (FIG. 8 ).

Example 2: Neutralization of SARS-CoV-2 BA.1 Omicron Lineage (a.k.a.B.1.1.529) Pseudovirus by BNT162b2 Vaccine-Elicited Human Sera

Materials and Methods:

A recombinant replication-deficient VSV vector that encodes greenfluorescent protein (GFP) and luciferase (Luc) instead of theVSV-glycoprotein (VSV-G) was pseudotyped with Wuhan-Hu-1 isolateSARS-CoV-2 spike (S) (GenBank: QHD43416.1), and a variant spikeharbouring the mutations found in the S protein of the Omicron(B.1.1.529) BA.1 lineage (A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211,L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S,S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G,H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F) accordingto published pseudotyping protocols. In brief, HEK293T/17 monolayerstransfected to express the respective SARS-CoV-2 S protein truncated ofthe C-terminal cytoplasmic 19 amino acids (SARS-CoV-2-S(CΔ19)) wereinoculated with VSVΔG-GFP/Luc vector. After incubation for 2 h at 37°C., the inoculum was removed, and cells were washed with PBS beforemedium supplemented with anti-VSV-G antibody (clone 8G5F11, Kerafast)was added to neutralise residual input virus. VSV-SARS-CoV-2pseudovirus-containing medium was collected 20 h after inoculation,0.2-μm-filtered and stored at −80° C.

For pseudovirus neutralisation assays, 40,000 Vero 76 cells were seededper 96-well. Sera were serially diluted 1:2 in culture medium startingwith a 1:10 dilution (dilution range of 1:10 to 1:10,240).VSV-SARS-CoV-2-S pseudoparticles were diluted in culture medium for afluorescent focus unit (ffu) count in the assay of ˜200 TU in the assay.Serum dilutions were mixed 1:1 with pseudovirus for 30 minutes at roomtemperature prior to addition to Vero 76 cell monolayers in 96-wellplates and incubation at 37° C. for 16-24 hours. Supernatants wereremoved, and the cells were lysed with luciferase reagent (Promega).Luminescence was recorded, and neutralisation titers were calculated asthe reciprocal of the highest serum dilution that still resulted in 50%reduction in luminescence. Results were reported as GMT of duplicates.If no neutralization was observed, an arbitrary titer value of 5 (halfof the limit of detection [LOD]) was reported.

Sera (N=19-20) were collected from subjects 21 days after receiving thesecond 30 μg dose or one month after receiving the third 30 μg dose ofBNT162b2. Each serum was tested for its neutralizing antibody titeragainst wild-type SARS-CoV-2 Wuhan Hu-1 and Omicron BA.1 lineage(B.1.1.529) spike protein pseudotyped VSV by a 50% neutralization assay(pVNT₅₀). The Omicron BA.1-strain spike protein used in theneutralization assay carried the following amino acid changes comparedto the Wuhan reference: A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211,L212I, Ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S,S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G,H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F.

BNT162b2-immune sera generated at 21 days after the second dosedisplayed effective neutralization of the SARS-CoV-2 Wuhan Hu-1pseudotyped reference. However, more than a 25-fold reduction inneutralization titers against the Omicron BA.1 variant was observed whencompared to the Wuhan reference (geometric mean titer [GMT] of 6 vs.155). Importantly, the third dose significantly increased theneutralizing antibody titers against the Omicron BA.1 strain pseudovirusby 25-fold. Hence, neutralization titers against the Omicron BA.1variant pseudovirus after three doses of BNT162b2 were comparable to theneutralization titers against the wild-type strain observed in sera fromindividuals who received two doses of BNT162b2 (GMT of 154 vs. 155).

Example 3: Additional Data for Neutralization of SARS-CoV-2 Omicron BA.1Lineage (a.k.a. B.1.1.529) Pseudovirus by BNT162b2 Vaccine-ElicitedHuman Sera

Further to the study and data as described in Example 2, a longitudinalanalysis of neutralizing titers was also performed in an independentsmaller subset of subjects. Sera drawn at 21 days after dose 2 exhibiteda 19.6-fold reduction in GMT against the Omicron BA.1 variant comparedto the Wuhan reference pseudovirus (FIG. 12 ; GMT of 6 vs. 118). Serumobtained from study participants just prior to receiving the third doseof BNT162b2 (at a median 251 days following dose 2) had considerablyreduced neutralizing titers against the Wuhan pseudovirus (GMT of 14)while the Omicron BA.1-specific titers were below the limit ofdetection. The third dose of BNT162b2 resulted in a significant increasein neutralizing titers against the Wuhan pseudovirus (GMT of 254) anda >26.6-fold increase in neutralizing titers against Omicron BA.1 at 1month after dose 3 compared to titers at 21 days after dose 2 (GMT of160 vs. 6). In all 9 subjects reduced but effective neutralization ofOmicron BA.1 was observed up to 3 months after the third dose (3.2-foldreduction compared to 1 month after dose 3; GMT of 50 vs. 160), whereasWuhan-specific neutralizing GMTs remained stable.

In summary, a third dose of BNT162b2 boosts Omicron BA.1 neutralizationcapability to a level similar to the one observed after two dosesagainst the Wuhan pseudovirus. Thus, the data indicate that providing athird dose of BNT162b2 can improve protection against infection with theOmicron BA.1 variant.

Example 4: Neutralization of Other SARS-CoV-2 Lineage Pseudovirus byBNT162b2 Vaccine-Elicited Human Sera

As described in Example 2 and Example 3, each serum was also tested forits neutralizing antibody titer against Beta and Delta lineage spikeprotein pseudotyped VSV by a 50% neutralization assay (pVNT₅₀) (data notshown).

A recombinant replication-deficient VSV vector that encodes greenfluorescent protein (GFP) and luciferase (Luc) instead of theVSV-glycoprotein (VSV-G) was pseudotyped with Wuhan-Hu-1 isolateSARS-CoV-2 spike (S) (GenBank: QHD43416.1), and a variant spikeharbouring the mutations found in the S protein of the Beta lineage(mutations: L18F, D80A, D215G, R246I, Δ242-244, K417N, E484K, N501Y,D614G, A701V), and the Delta lineage (mutations: T19R, G142D, Δ157/158,K417N, L452R, T478K, D614G, P681R, D950N, K986P, V987P), according topublished pseudotyping protocols.

For sera collected 21 days after a second dose of BNT162b2, PVNT₅₀ wasreduced by approximately 6.7-fold (GMT of 24 vs 155) for the Betavariant and approximately 2.2-fold for the Delta variant (GMT of 73 vs155) as compared to the Wuhan variant, but were significantly higherthan the neutralization response against the delta variant. The thirddose of BNT162b2 also increased neutralizing activity against Beta andDelta pseudoviruses, with GMTs of 279 and 413, respectively.

Example 5: T Cell Epitope Conservation in the Omicron BA.1 Spike Variant

In addition to humoral immunity, T-cell mediated immunity is anotherlayer of defense, in particular for preventing severe COVID-19. Previousobservations that efficacy against disease is already established about12 days after the first dose of BNT162b2 before the second dose has beenadministered and prior to the onset of high neutralizing titers furtherhighlights the potential protective role of the T cell response. Priorreports have shown that CD8 T cell responses in individuals vaccinatedwith BNT162b2 are polyepitopic.

To assess the risk of immune evasion of CD8+ T cell responses by OmicronBA.1, a set of HLA class I restricted T cell epitopes from the Wuhanspike protein sequence that were reported in the Immune Epitope Databaseto be immunogenic (IEDB, n=244) were investigated (the procedure used toidentify these epitopes is described in the below paragraph). Despitethe multitude of mutations in the Omicron BA.1 spike protein, 85.25%(n=208) of the described epitopes were not impacted on the amino acidsequence level, indicating that the targets of the vast majority of Tcell responses elicited by BNT162b2 may still be conserved in theOmicron BA.1 variant (FIG. 13 ). Early laboratory studies confirm thatCD8+ T cell recognition of Omicron epitopes are preserved in COVID-19recovered individuals exposed early in the pandemic and that the OmicronBA.1 VOC has not evolved extensive T-cell escape mutations at this time.

To estimate the rate of nonsynonymous mutation in T cell epitopes in thespike glycoprotein, the Immune Epitope Database (https://www.iedb.org/)was used to obtain epitopes confirmed for T cell reactivity inexperimental assays. The database was filtered using the followingcriteria: Organism: SARS-COV2; Antigen: Spike glycoprotein; PositiveAssay; No B cell assays; No MHC assays; MHC Restriction Type: Class I;Host: Homo sapiens (human). The resulting table was filtered by removingepitopes that were “deduced from a reactive overlapping peptide pool”,as well as epitopes longer than 14 amino acids in order to restrict thedataset to confirmed minimal epitopes only. Of the 251 unique epitopesequences obtained in this approach, 244 were found in the Wuhan strainSpike glycoprotein. Of these, 36 epitopes (14.75%) included a positionreported to be mutated in Omicron by the sequence analysis disclosedherein. Results are summarized in FIG. 10 . Also shown are the numbersof predicted MHC-I epitopes mutated in each of the Alpha, Beta, Gamma,Delta SARS-CoV-2 variants. FIG. 13 depicts the locations of the T cellepitopes within the Spike Protein, and indicates which epitopes areconserved or mutated in the Spike protein from the Omicron BA.1 variant.

Example 6: Exemplary Dosing Regimens

In some embodiments, compositions and methods disclosed herein can beused in accordance with an exemplary vaccination regimen as illustratedin FIG. 14 .

Primary Dosing Regimens

In some embodiments, subjects are administered a primary dosing regimen.A primary dosing regimen can comprise one or more doses. For example, insome embodiments, a primary dosing regimen comprises a single dose(PD₁). In some embodiments a primary dosing regimen comprises a firstdose (PD₁) and a second dose (PD₂). In some embodiments, a primarydosing regimen comprises a first dose, a second dose, and a third dose(PD₃). In some embodiments, a primary dosing regimen comprises a firstdose, a second dose, a third dose, and one or more additional doses(PD_(n)) of any one of the pharmaceutical compositions described herein.

In some embodiments, PD₁ comprises administering 1 to 100 ug of RNA. Insome embodiments, PD₁ comprises administering 1 to 60 ug of RNA In someembodiments, PD₁ comprises administering 1 to 50 ug of RNA. In someembodiments, PD₁ comprises administering 1 to 30 ug of RNA. In someembodiments, PD₁ comprises administering about 3 ug of RNA. In someembodiments, PD₁ comprises administering about 5 ug of RNA. In someembodiments, PD₁ comprises administering about 10 ug of RNA. In someembodiments, PD₁ comprises administering about 15 ug of RNA. In someembodiments, PD₁ comprises administering about 20 ug of RNA. In someembodiments, PD₁ comprises administering about ug of RNA. In someembodiments, PD₁ comprises administering about 50 ug of RNA. In someembodiments, PD₁ comprises administering about 60 ug of RNA.

In some embodiments, PD₂ comprises administering 1 to 100 ug of RNA. Insome embodiments, PD₂ comprises administering 1 to 60 ug of RNA. In someembodiments, PD₂ comprises administering 1 to 50 ug of RNA. In someembodiments, PD₂ comprises administering 1 to 30 ug of RNA. In someembodiments, PD₂ comprises administering about 3 ug. In someembodiments, PD₂ comprises administering about 5 ug of RNA. In someembodiments, PD₂ comprises administering about 10 ug of RNA. In someembodiments, PD₂ comprises administering about 15 ug of RNA. In someembodiments, PD₂ comprises administering about 20 ug RNA. In someembodiments, PD₂ comprises administering about ug of RNA. In someembodiments, PD₂ comprises administering about 50 ug of RNA. In someembodiments, PD₂ comprises administering about 60 ug of RNA.

In some embodiments, PD₃ comprises administering 1 to 100 ug of RNA. Insome embodiments, PD₃ comprises administering 1 to 60 ug of RNA. In someembodiments, PD₃ comprises administering 1 to 50 ug of RNA. In someembodiments, PD₃ comprises administering 1 to 30 ug of RNA. In someembodiments, PD₃ comprises administering about 3 ug of RNA. In someembodiments, PD₃ comprises administering about 5 ug of RNA. In someembodiments, PD₃ comprises administering about 10 ug of RNA. In someembodiments, PD₃ comprises administering about 15 ug of RNA. In someembodiments, PD₃ comprises administering about 20 ug of RNA. In someembodiments, PD₃ comprises administering about ug of RNA. In someembodiments, PD₃ comprises administering about 50 ug of RNA. In someembodiments, PD₃ comprises administering about 60 ug of RNA.

In some embodiments, PD_(n) comprises administering 1 to 100 ug of RNA.In some embodiments, PD_(n) comprises administering 1 to 60 ug of RNA.In some embodiments, PD_(n) comprises administering 1 to 50 ug of RNA.In some embodiments, PD_(n) comprises administering 1 to 30 ug of RNA.In some embodiments, PD_(n) comprises administering about 3 ug of RNA.In some embodiments, PD_(n) comprises administering about 5 ug of RNA.In some embodiments, PD_(n) comprises administering about 10 ug of RNA.In some embodiments, PD_(n) comprises administering about 15 ug of RNA.In some embodiments, PD_(n) comprises administering about 20 ug of RNA.In some embodiments, PD_(n) comprises administering about ug of RNA. Insome embodiments, PD_(n) comprises administering about 50 ug of RNA. Insome embodiments, PD_(n) comprises administering about 60 ug of RNA.

In some embodiments, PD₁ comprises an RNA encoding a SARS-CoV-2 Spikeprotein or an immunogenic fragment thereof from the Wuhan strain. Insome embodiments, PD₁ comprises an RNA encoding a Spike protein or animmunogenic fragment thereof from a SARS-CoV-2 strain that is prevalentand/or spreading rapidly in a relevant jurisdiction. In someembodiments, PD₁ comprises an RNA encoding a SARS-CoV-2 Spike protein oran immunogenic fragment thereof comprising one or more mutations from analpha variant. In some embodiments, PD₁ comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof comprisingone or more mutations from a delta variant. In some embodiments, PD₁comprises an RNA encoding a SARS-CoV-2 Spike protein or an immunogenicfragment thereof comprising one or more mutations from a beta variant.In some embodiments, PD₁ comprises an RNA encoding a SARS-CoV-2 Spikeprotein or an immunogenic fragment thereof comprising one or moremutations from an Omicron variant (e.g., a BA.4/5, BA.1, BA.2, XBB,XBB.1, or BQ.1 Omicron variant). In some embodiments, PD₁ comprises anRNA encoding a SARS-CoV-2 Spike protein or an immunogenic fragmentthereof from the Wuhan strain and one or more additional RNAs encoding aSpike protein or an immunogenic fragment thereof from a SARS-CoV-2strain that is prevalent and/or spreading rapidly in a relevantjurisdiction. In some embodiments, PD₁ comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from analpha variant. In some embodiments, PD₁ comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from abeta variant. In some embodiments, PD₁ comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from adelta variant. In some embodiments, PD₁ comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from anOmicron variant (e.g., a BA.4/5, BA.1, BA.2, XBB, XBB.1, or BQ.1 Omicronvariant).

In some embodiments, PD₂ comprises an RNA encoding a SARS-CoV-2 Spikeprotein or an immunogenic fragment thereof from the Wuhan strain. Insome embodiments, PD₂ comprises an RNA encoding a Spike protein or animmunogenic fragment thereof from a SARS-CoV-2 strain that is prevalentand/or spreading rapidly in a relevant jurisdiction. In someembodiments, PD₂ comprises an RNA encoding a SARS-CoV-2 Spike protein oran immunogenic fragment thereof comprising one or more mutations from analpha variant. In some embodiments, PD₂ comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof comprisingone or more mutations from a delta variant. In some embodiments, PD₂comprises an RNA encoding a SARS-CoV-2 Spike protein or an immunogenicfragment thereof comprising one or more mutations from a beta variant.In some embodiments, PD₂ comprises an RNA encoding a SARS-CoV-2 Spikeprotein or an immunogenic fragment thereof comprising one or moremutations from an Omicron variant (e.g., a BA.4/5, BA.1, BA.2, XBB,XBB.1, or BQ.1 Omicron variant). In some embodiments, PD₂ comprises anRNA encoding a SARS-CoV-2 Spike protein or an immunogenic fragmentthereof from the Wuhan strain and one or more additional RNAs encoding aSpike protein or an immunogenic fragment thereof from a SARS-CoV-2strain that is prevalent and/or spreading rapidly in a relevantjurisdiction. In some embodiments, PD₂ comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from analpha variant. In some embodiments, PD₂ comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from abeta variant. In some embodiments, PD₂ comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from adelta variant. In some embodiments, PD₂ comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from anOmicron variant (e.g., a BA.4/5, BA.1, BA.2, XBB, XBB.1, or BQ.1 Omicronvariant).

In some embodiments, PD₃ comprises an RNA encoding a SARS-CoV-2 Spikeprotein or an immunogenic fragment thereof from the Wuhan strain. Insome embodiments, PD₃ comprises an RNA encoding a Spike protein or animmunogenic fragment thereof from a SARS-CoV-2 strain that is prevalentand/or spreading rapidly in a relevant jurisdiction. In someembodiments, PD₃ comprises an RNA encoding a SARS-CoV-2 Spike protein oran immunogenic fragment thereof comprising one or more mutations from analpha variant. In some embodiments, PD₃ comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof comprisingone or more mutations from a delta variant. In some embodiments, PD₃comprises an RNA encoding a SARS-CoV-2 Spike protein or an immunogenicfragment thereof comprising one or more mutations from a beta variant.In some embodiments, PD₃ comprises an RNA encoding a SARS-CoV-2 Spikeprotein or an immunogenic fragment thereof comprising one or moremutations from an Omicron variant (e.g., a BA.4/5, BA.1, BA.2, XBB,XBB.1, or BQ.1 Omicron variant). In some embodiments, PD₃ comprises anRNA encoding a SARS-CoV-2 Spike protein or an immunogenic fragmentthereof from the Wuhan strain and one or more additional RNAs encoding aSpike protein or an immunogenic fragment thereof from a SARS-CoV-2strain that is prevalent and/or spreading rapidly in a relevantjurisdiction. In some embodiments, PD₃ comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from analpha variant. In some embodiments, PD₃ comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from abeta variant. In some embodiments, PD₃ comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from adelta variant. In some embodiments, PD₃ comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from anOmicron variant (e.g., a BA.4/5, BA.1, BA.2, XBB, XBB.1, or BQ.1 Omicronvariant).

In some embodiments, PD_(n) comprises an RNA encoding a SARS-CoV-2 Spikeprotein or an immunogenic fragment thereof from the Wuhan strain. Insome embodiments, PD_(n) comprises an RNA encoding a Spike protein or animmunogenic fragment thereof from a SARS-CoV-2 strain that is prevalentand/or spreading rapidly in a relevant jurisdiction. In someembodiments, PD_(n) comprises an RNA encoding a SARS-CoV-2 Spike proteinor an immunogenic fragment thereof comprising one or more mutations froman alpha variant. In some embodiments, PD_(n) comprises an RNA encodinga SARS-CoV-2 Spike protein or an immunogenic fragment thereof comprisingone or more mutations from a delta variant. In some embodiments, PD_(n)comprises an RNA encoding a SARS-CoV-2 Spike protein or an immunogenicfragment thereof comprising one or more mutations from a beta variant.In some embodiments, PD_(n) comprises an RNA encoding a SARS-CoV-2 Spikeprotein or an immunogenic fragment thereof comprising one or moremutations from an Omicron variant (e.g., a BA.4/5, BA.1, BA.2, XBB,XBB.1, or BQ.1 Omicron variant). In some embodiments, PD_(n) comprisesan RNA encoding a SARS-CoV-2 Spike protein or an immunogenic fragmentthereof from the Wuhan strain and one or more additional RNAs encoding aSpike protein or an immunogenic fragment thereof from a SARS-CoV-2strain that is prevalent and/or spreading rapidly in a relevantjurisdiction. In some embodiments, PD_(n) comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from analpha variant. In some embodiments, PD_(n) comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from abeta variant. In some embodiments, PD_(n) comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from adelta variant. In some embodiments, PD_(n) comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from anOmicron variant (e.g., a BA.4/5, BA.1, BA.2, XBB, XBB.1, or BQ.1 Omicronvariant).

In some embodiments, PD₁, PD₂, PD₃, and PD_(n) can each independentlycomprise a plurality of (e.g., at least two) mRNA compositions describedherein. In some embodiments PD₁, PD₂, PD₃, and PD_(n) can eachindependently comprise a first and a second mRNA composition. In someembodiments, at least one of a plurality of mRNA compositions comprisesBNT162b2 (e.g., as described herein). In some embodiments, at least oneof a plurality of mRNA compositions comprises an mRNA encoding aSARS-CoV-2 S protein or an immunogenic fragment thereof from a differentSARS-CoV-2 variant. In some embodiments, at least one of a plurality ofmRNA compositions comprises an mRNA encoding a SARS-CoV-2 S protein oran immunogenic fragment thereof from a Wuhan strain of SARS-CoV-2. Insome embodiments, at least one of a plurality of mRNA compositionscomprises an RNA encoding a SARS-CoV-2 S protein or an immunogenicfragment thereof comprising one or more mutations from a variant that isprevalent and/or spreading rapidly in a relevant jurisdiction. In someembodiments, at least one of a plurality of mRNA compositions comprisesan mRNA encoding a SARS-CoV-2 S protein or an immunogenic fragmentthereof comprising one or more mutations from an alpha variant. In someembodiments, at least one of a plurality of mRNA compositions comprisesan mRNA encoding a SARS-CoV-2 S protein or an immunogenic fragmentthereof comprising one or more mutations from a delta variant. In someembodiments, at least one of a plurality of mRNA compositions comprisesan mRNA encoding a SARS-CoV-2 S protein or an immunogenic fragmentthereof comprising one or more mutations from an Omicron variant (e.g.,a BA.4/5, BA.1, BA.2, XBB, XBB.1, or BQ.1 Omicron variant).

In some embodiments, a plurality of mRNA compositions given in PD₁, PD₂,PD₃ and/or PD_(n) can each independently comprise at least two differentmRNA constructs (e.g., differing in at protein-encoding sequences). Forexample, in some embodiments a plurality of mRNA compositions given inPD₁, PD₂, PD₃, and/or PD_(n) can each independently comprise an mRNAencoding a SARS-CoV-2 S protein or an immunogenic fragment thereof froma Wuhan strain of SARS-CoV-2 and an mRNA encoding a SARS-CoV-2 S proteinor an immunogenic fragment thereof comprising one or more mutations froma variant that is prevalent and/or spreading rapidly in a relevantjurisdiction. In some embodiments a plurality of mRNA compositions givenin PD₁, PD₂, PD₃, and/or PD_(n) can each independently comprise an mRNAencoding a SARS-CoV-2 S protein or an immunogenic fragment thereofderived from a Wuhan strain of SARS-CoV-2 and an mRNA encoding aSARS-CoV-2 S protein or an immunogenic fragment thereof comprising oneor more mutations from a variant that is prevalent and/or spreadingrapidly in a relevant jurisdiction. In some such embodiments, a variantcan be an alpha variant. In some such embodiments, a variant can be adelta variant. In some such embodiments a variant can be an Omicronvariant (e.g., a BA.4/5, BA.1, BA.2, XBB, XBB.1, or BQ.1 Omicronvariant). In some embodiments, each of a plurality of mRNA compositionsgiven in PD₁, PD₂, PD₃, and/or PD_(n) can independently comprise atleast two mRNAs, each encoding a SARS-CoV-2 S protein or an immunogenicfragment thereof comprising one or more mutations from a distinctvariant that is prevalent and/or spreading rapidly in a relevantjurisdiction. In some embodiments, each of a plurality of mRNAcompositions given in PD₁, PD₂, PD₃, and/or PD_(n) can independentlycomprise an mRNA encoding a SARS-CoV-2 S protein or an immunogenicfragment thereof from an alpha variant and an mRNA encoding a SARS-CoV-2S protein or an immunogenic fragment thereof comprising one or moremutations from a delta variant. In some embodiments, each of a pluralityof mRNA compositions given in PD₁, PD₂, PD₃, and/or PD_(n) canindependently comprise an mRNA encoding a SARS-CoV-2 S protein or animmunogenic fragment thereof from an alpha variant and an mRNA encodinga SARS-CoV-2 S protein or an immunogenic fragment thereof comprising oneor more mutations from an Omicron variant (e.g., a BA.4/5, BA.1, BA.2,XBB, XBB.1, or BQ.1 Omicron variant). In some embodiments, each of aplurality of mRNA compositions given in PD₁, PD₂, PD₃, and/or PD_(n) canindependently comprise an mRNA encoding a SARS-CoV-2 S protein or animmunogenic fragment thereof from a delta variant and an mRNA encoding aSARS-CoV-2 S protein or an immunogenic fragment thereof comprising oneor more mutations from an Omicron variant (e.g., a BA.4/5, BA.1, BA.2,XBB, XBB.1, or BQ.1 Omicron variant).

In some embodiments, PD₁, PD₂, PD₃, and/or PD_(n) each comprise aplurality of mRNA compositions, wherein each mRNA composition isseparately administered to a subject. For example, in some embodimentseach mRNA composition is administered via intramuscular injection atdifferent injection sites. For example, in some embodiments, a first andsecond mRNA composition given in PD₁, PD₂, PD₃, and/or PD_(n) areseparately administered to different arms of a subject via intramuscularinjection.

In some embodiments, PD₁, PD₂, PD₃, and/or PD_(n) comprise administeringa plurality of RNA molecules, wherein each RNA molecule encodes a Spikeprotein comprising mutations from a different SARS-CoV-2 variant, andwherein the plurality of RNA molecules are administered to the subjectin a single formulation. In some embodiments, the single formulationcomprises an RNA encoding a Spike protein or an immunogenic variantthereof from the Wuhan strain and an RNA encoding a SARS-CoV-2 Spikeprotein or an immunogenic fragment thereof comprising one or moremutations from an alpha variant. In some embodiments, the singleformulation comprises an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof from the Wuhan strain and an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof comprisingone or more mutations from a beta variant. In some embodiments, thesingle formulation comprises an RNA encoding a SARS-CoV-2 Spike proteinor an immunogenic fragment thereof from the Wuhan strain and an RNAencoding a SARS-CoV-2 Spike protein or an immunogenic fragment thereofcomprising one or more mutations from a delta variant. In someembodiments, the single formulation comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from anOmicron variant (e.g., a BA.4/5, BA.1, BA.2, XBB, XBB.1, or BQ.1 Omicronvariant).

In some embodiments, the length of time between PD₁ and PD₂ (PI₁) is atleast about 1 week, at least about 2 weeks, at least about 3 weeks, orat least about 4 weeks. In some embodiments, PI₁ is about 1 week toabout 12 weeks. In some embodiments, PI₁ is about 1 week to about 10weeks. In some embodiments, PI₁ is about 2 weeks to about 10 weeks. Insome embodiments, PI₁ is about 2 weeks to about 8 weeks. In someembodiments, PI₁ is about 3 weeks to about 8 weeks. In some embodiments,PI₁ is about 4 weeks to about 8 weeks. In some embodiments, PI₁ is about6 weeks to about 8 weeks. In some embodiments PI₁ is about 3 to about 4weeks. In some embodiments, PI₁ is about 1 week. In some embodiments,PI₁ is about 2 weeks. In some embodiments, PI₁ is about 3 weeks. In someembodiments, PI₁ is about 4 weeks. In some embodiments, PI₁ is about 5weeks. In some embodiments, PI₁ is about 6 weeks. In some embodiments,PI₁ is about 7 weeks. In some embodiments, PI₁ is about 8 weeks. In someembodiments, PI₁ is about 9 weeks. In some embodiments, PI₁ is about 10weeks. In some embodiments, PI₁ is about 11 weeks. In some embodiments,PI₁ is about 12 weeks.

In some embodiments, the length of time between PD₂ and PD₃ (PI₂) is atleast about 1 week, at least about 2 weeks, or at least about 3 weeks.In some embodiments, PI₂ is about 1 week to about 12 weeks. In someembodiments, PI₂ is about 1 week to about 10 weeks. In some embodiments,PI₂ is about 2 weeks to about 10 weeks. In some embodiments, PI₂ isabout 2 weeks to about 8 weeks. In some embodiments, PI₂ is about 3weeks to about 8 weeks. In some embodiments, PI₂ is about 4 weeks toabout 8 weeks. In some embodiments, PI₂ is about 6 weeks to about 8weeks. In some embodiments PI₂ is about 3 to about 4 weeks. In someembodiments, PI₂ is about 1 week. In some embodiments, PI₂ is about 2weeks. In some embodiments, PI₂ is about 3 weeks. In some embodiments,PI₂ is about 4 weeks. In some embodiments, PI₂ is about 5 weeks. In someembodiments, PI₂ is about 6 weeks. In some embodiments, PI₂ is about 7weeks. In some embodiments, PI₂ is about 8 weeks. In some embodiments,PI₂ is about 9 weeks. In some embodiments, PI₂ is about 10 weeks. Insome embodiments, PI₂ is about 11 weeks. In some embodiments, PI₂ isabout 12 weeks.

In some embodiments, the length of time between PD₃ and a subsequentdose that is part of the Primary Dosing Regimen, or between doses forany dose beyond PD₃ (PI_(n)) is each separately and independentlyselected from: about 1 week or more, about 2 weeks or more, or about 3weeks or more. In some embodiments, PI_(n) is about 1 week to about 12weeks. In some embodiments, PI_(n) is about 1 week to about 10 weeks. Insome embodiments, PI_(n) is about 2 weeks to about 10 weeks. In someembodiments, PI_(n) is about 2 weeks to about 8 weeks. In someembodiments, PI_(n) is about 3 weeks to about 8 weeks. In someembodiments, PI_(n) is about 4 weeks to about 8 weeks. In someembodiments, PI_(n) is about 6 weeks to about 8 weeks. In someembodiments PI_(n) is about 3 to about 4 weeks. In some embodiments, PI₂is about 1 week. In some embodiments, PI_(n) is about 2 weeks. In someembodiments, PI_(n) is about 3 weeks. In some embodiments, PI_(n) isabout 4 weeks. In some embodiments, PI_(n) is about 5 weeks. In someembodiments, PI_(n) is about 6 weeks. In some embodiments, PI_(n) isabout 7 weeks. In some embodiments, PI_(n) is about 8 weeks. In someembodiments, PI_(n) is about 9 weeks. In some embodiments, PI_(n) isabout 10 weeks. In some embodiments, PI_(n) is about 11 weeks. In someembodiments, PI_(n) is about 12 weeks.

In some embodiments, one or more compositions adminstered in PD₁ areformulated in a Tris buffer. In some embodiments, one or morecompositions administered in PD₂ are formulated in a Tris buffer. Insome embodiments, one or more compositions administering in PD₃ areformulated in a Tris buffer. In some embodiments, one or morecompositions administered in PD_(n) are formulated in a Tris buffer.

In some embodiments, the primary dosing regimen comprises administeringtwo or more mRNA compositions described herein, and at least two of themRNA compositions have different formulations. In some embodiments, theprimary dosing regimen comprises PD₁ and PD₂, where PD₁ comprisesadministering an mRNA formulated in a Tris buffer and PD₂ comprisesadministering an mRNA formulated in a PBS buffer. In some embodiments,the primary dosing regimen comprises PD₁ and PD₂, where PD₁ comprisesadministering an mRNA formulated in a PBS buffer and PD₂ comprisesadministering an mRNA formulated in a Tris buffer.

In some embodiments, one or more mRNA compositions given in PD₁, PD₂,PD₃, and/or PD_(n) can be administered in combination with anothervaccine. In some embodiments, another vaccine is for a disease that isnot COVID-19. In some embodiments, the disease is one that increasesdeleterious effects of SARS-CoV-2 when a subject is coinfected with thedisease and SARS-CoV-2. In some embodiments, the disease is one thatincreases the transmission rate of SARS-CoV-2 when a subject iscoinfected with the disease and SARS-CoV-2. In some embodiments, anothervaccine is a different commerically available vaccine. In someembodiments, the different commercially available vaccine is an RNAvaccine. In some embodiments, the different commercially availablevaccine is a polypeptide-based vaccine. In some embodiments, anothervaccine (e.g., as described herein) and one or more mRNA compositionsgiven in PD₁, PD₂, PD₃, and/or PD_(n) are separately administered, forexample, in some embodiments via intramuscular injection, at differentinjection sites. For example, in some embodiments, an influenza vaccineand one or more SARS-CoV-2 mRNA compositions described herein given inPD₁, PD₂, PD₃, and/or PD_(n) are separately administered to differentarms of a subject via intramuscular injection.

Booster Dosing Regimens

In some embodiments, methods of vaccination disclosed herein compriseone or more Booster Dosing Regimens. The Booster Dosing Regimensdisclosed herein comprise one or more doses. In some embodiments, aBooster Dosing Regimen is administered to patients who have beenadministered a Primary Dosing Regimen (e.g., as described herein). Insome embodiments a Booster Dosing Regimen is administered to patientswho have not received a pharmaceutical composition disclosed herein. Insome embodiments a Booster Dosing Regimen is administered to patientswho have been previously vaccinated with a COVID-19 vaccine that isdifferent from the vaccine administered in a Primary Dosing Regimen.

In some embodiments, the length of time between the Primary DosingRegimen and the Booster Dosing Regimen is at least 1 week, at least 2weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at leastweeks, at least 11 weeks, at least 12 weeks, at least 2 months, at least3 months, at least 4 months, at least 5 months, at least 6 months, atleast 7 months, at least 8 months, at least 9 months, at least 10months, at least 11 months, or at least 12 months or longer. In someembodiments, the length of time between the Primary Dosing Regimen andthe Booster Dosing Regimen is about 1 month. In some embodiments, thelength of time between the Primary Dosing Regimen and the Booster DosingRegimen is at least about 2 months. In some embodiments, the length oftime between the Primary Dosing Regimen and the Booster Dosing Regimenis at least about 3 months. In some embodiments, the length of timebetween the Primary Dosing Regimen and the Booster Dosing Regimen is atleast about 4 months. In some embodiments, the length of time betweenthe Primary Dosing Regimen and the Booster Dosing Regimen is at leastabout 5 months. In some embodiments, the length of time between thePrimary Dosing Regimen and the Booster Dosing Regimen is at least about6 months. In some embodiments, the length of time between the PrimaryDosing Regimen and the Booster Dosing Regimen is from about 1 month toabout 48 months. In some embodiments, the length of time between thePrimary Dosing Regimen and the Booster Dosing Regimen is from about 1month to about 36 months. In some embodiments, the length of timebetween the primary dosing regimen and the Booster Dosing Regimen isfrom about 1 month to about 24 months. In some embodiments, the lengthof time between the Primary Dosing Regimen and the Booster DosingRegimen is from about 2 months to about 24 months. In some embodiments,the length of time between the Primary Dosing Regimen and the BoosterDosing Regimen is from about 3 months to about 24 months. In someembodiments, the length of time between the primary dosing regimen andthe Booster Dosing Regimen is from about 3 months to about 18 months. Insome embodiments, the length of time between the primary dosing regimenand the Booster Dosing Regimen is from about 3 months to about 12months. In some embodiments, the length of time between the primarydosing regimen and the Booster Dosing Regimen is from about 6 months toabout 12 months. In some embodiments, the length of time between thePrimary Dosing Regimen and the Booster Dosing Regimen is from about 3months to about 9 months. In some embodiments, the length of timebetween the Primary Dosing Regimen and the Booster Dosing Regimen isfrom about 5 months to about 7 months. In some embodiments, the lengthof time between the Primary Dosing Regimen and the Booster DosingRegimen is about 6 months.

In some embodiments, subjects are administered a Booster Dosing Regimen.A Booster dosing regimen can comprise one or more doses. For example, insome embodiments, a Booster Dosing Regimen comprises a single dose(BD₁). In some embodiments a Booster Dosing Regimen comprises a firstdose (BD₁) and a second dose (BD₂). In some embodiments, a BoosterDosing Regimen comprises a first dose, a second dose, and a third dose(BD₃). In some embodiments, a Booster Dosing Regimen comprises a firstdose, a second dose, a third dose, and one or more additional doses(BD_(n)) of any one of the pharmaceutical compositions described herein.

In some embodiments, BD₁ comprises administering 1 to 100 ug of RNA. Insome embodiments, BD₁ comprises administering 1 to 60 ug of RNA. In someembodiments, BD₁ comprises administering 1 to 50 ug of RNA. In someembodiments, BD₁ comprises administering 1 to 30 ug of RNA. In someembodiments, BD₁ comprises administering about 3 ug of RNA. In someembodiments, BD₁ comprises administering about 5 ug of RNA. In someembodiments, BD₁ comprises administering about 10 ug of RNA. In someembodiments, BD, comprises administering about 15 ug of RNA. In someembodiments, BD₁ comprises administering about 20 ug of RNA. In someembodiments, BD₁ comprises administering about ug of RNA. In someembodiments, BD₁ comprises administering about 50 ug of RNA. In someembodiments, BD₁ comprises administering about 60 ug of RNA.

In some embodiments, BD₂ comprises administering 1 to 100 ug of RNA. Insome embodiments, BD₂ comprises administering 1 to 60 ug of RNA. In someembodiments, BD₂ comprises administering 1 to 50 ug of RNA. In someembodiments, BD₂ comprises administering 1 to 30 ug of RNA. In someembodiments, BD₂ comprises administering about 3 ug. In someembodiments, BD₂ comprises administering about 5 ug of RNA. In someembodiments, BD₂ comprises administering about 10 ug of RNA. In someembodiments, BD₂ comprises administering about 15 ug of RNA. In someembodiments, BD₂ comprises administering about 20 ug RNA. In someembodiments, BD₂ comprises administering about ug of RNA. In someembodiments, BD₂ comprises administering about 50 ug of RNA. In someembodiments, BD₂ comprises administering about 60 ug of RNA.

In some embodiments, BD₃ comprises administering 1 to 100 ug of RNA. Insome embodiments, BD₃ comprises administering 1 to 60 ug of RNA. In someembodiments, BD₃ comprises administering 1 to 50 ug of RNA. In someembodiments, BD₃ comprises administering 1 to 30 ug of RNA. In someembodiments, BD₃ comprises administering about 3 ug of RNA. In someembodiments, BD₃ comprises administering about 5 ug of RNA. In someembodiments, BD₃ comprises administering about 10 ug of RNA. In someembodiments, BD₃ comprises administering about 15 ug of RNA. In someembodiments, BD₃ comprises administering about 20 ug of RNA. In someembodiments, BD₃ comprises administering about ug of RNA. In someembodiments, BD₃ comprises administering about 50 ug of RNA. In someembodiments, BD₃ comprises administering about 60 ug of RNA.

In some embodiments, BD_(n) comprises administering 1 to 100 ug of RNA.In some embodiments, BD_(n) comprises administering 1 to 60 ug of RNA.In some embodiments, BD_(n) comprises administering 1 to 50 ug of RNA.In some embodiments, BD_(n) comprises administering 1 to 30 ug of RNA.In some embodiments, BD_(n) comprises administering about 3 ug of RNA.In some embodiments, BD_(n) comprises administering about 5 ug of RNA.In some embodiments, BD_(n) comprises administering about 10 ug of RNA.In some embodiments, BD_(n) comprises administering about 15 ug of RNA.In some embodiments, BD_(n) comprises administering about 20 ug of RNA.In some embodiments, BD_(n) comprises administering about ug of RNA. Insome embodiments, BD_(n) comprises administering about 60 ug of RNA. Insome embodiments, BD_(n) comprises administering about 50 ug of RNA.

In some embodiments, BD₁ comprises an RNA encoding a SARS-CoV-2 Spikeprotein or an immunogenic fragment thereof from the Wuhan strain. Insome embodiments, BD₁ comprises an RNA encoding a Spike protein or animmunogenic fragment thereof from a SARS-CoV-2 strain that is prevalentand/or spreading rapidly in a relevant jurisdiction. In someembodiments, BD₁ comprises an RNA encoding a SARS-CoV-2 Spike protein oran immunogenic fragment thereof comprising one or more mutations from analpha variant. In some embodiments, BD₁ comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof comprisingone or more mutations from a delta variant. In some embodiments, BD₁comprises an RNA encoding a SARS-CoV-2 Spike protein or an immunogenicfragment thereof comprising one or more mutations from a beta variant.In some embodiments, BD₁ comprises an RNA encoding a SARS-CoV-2 Spikeprotein or an immunogenic fragment thereof comprising one or moremutations from an Omicron variant (e.g., a BA.4/5, BA.1, BA.2, XBB,XBB.1, or BQ.1 Omicron variant).

In some embodiments, BD₁ comprises an RNA encoding a SARS-CoV-2 Spikeprotein or an immunogenic fragment thereof from the Wuhan strain and oneor more RNA encoding a Spike protein or an immunogenic fragment thereoffrom a SARS-CoV-2 strain that is prevalent and/or spreading rapidly in arelevant jurisdiction. In some embodiments, BD₁ comprises an RNAencoding a SARS-CoV-2 Spike protein or an immunogenic fragment thereoffrom the Wuhan strain and an RNA encoding a SARS-CoV-2 Spike protein oran immunogenic fragment thereof comprising one or more mutations from aalpha variant. In some embodiments, BD₁ comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from adelta variant. In some embodiments, BD₁ comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from abeta variant. In some embodiments, BD₁ comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from anOmicron variant (e.g., a BA.4/5, BA.1, BA.2, XBB, XBB.1, or BQ.1 Omicronvariant).

In some embodiments, BD₂ comprises an RNA encoding a SARS-CoV-2 Spikeprotein or an immunogenic fragment thereof from the Wuhan strain. Insome embodiments, BD₂ comprises an RNA encoding a Spike protein or animmunogenic fragment thereof from a SARS-CoV-2 strain that is prevalentand/or spreading rapidly in a relevant jurisdiction. In someembodiments, BD₂ comprises an RNA encoding a SARS-CoV-2 Spike protein oran immunogenic fragment thereof comprising one or more mutations from analpha variant. In some embodiments, BD₂ comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof comprisingone or more mutations from a delta variant. In some embodiments, BD₂comprises an RNA encoding a SARS-CoV-2 Spike protein or an immunogenicfragment thereof comprising one or more mutations from a beta variant.In some embodiments, BD₂ comprises an RNA encoding a SARS-CoV-2 Spikeprotein or an immunogenic fragment thereof comprising one or moremutations from an Omicron variant (e.g., a BA.4/5, BA.1, BA.2, XBB,XBB.1, or BQ.1 Omicron variant). In some embodiments, BD₂ comprises anRNA encoding a SARS-CoV-2 Spike protein or an immunogenic fragmentthereof from the Wuhan strain and one or more RNA encoding a Spikeprotein or an immunogenic fragment thereof from a SARS-CoV-2 strain thatis prevalent and/or spreading rapidly in a relevant jurisdiction. Insome embodiments, BD₂ comprises an RNA encoding a SARS-CoV-2 Spikeprotein or an immunogenic fragment thereof from the Wuhan strain and anRNA encoding a SARS-CoV-2 Spike protein or an immunogenic fragmentthereof comprising one or more mutations from a alpha variant. In someembodiments, BD₂ comprises an RNA encoding a SARS-CoV-2 Spike protein oran immunogenic fragment thereof from the Wuhan strain and an RNAencoding a SARS-CoV-2 Spike protein or an immunogenic fragment thereofcomprising one or more mutations from a delta variant. In someembodiments, BD₂ comprises an RNA encoding a SARS-CoV-2 Spike protein oran immunogenic fragment thereof from the Wuhan strain and an RNAencoding a SARS-CoV-2 Spike protein or an immunogenic fragment thereofcomprising one or more mutations from a beta variant. In someembodiments, BD₂ comprises an RNA encoding a SARS-CoV-2 Spike protein oran immunogenic fragment thereof from the Wuhan strain and an RNAencoding a SARS-CoV-2 Spike protein or an immunogenic fragment thereofcomprising one or more mutations from an Omicron variant (e.g., aBA.4/5, BA.1, BA.2, XBB, XBB.1, or BQ.1 Omicron variant).

In some embodiments, BD₃ comprises an RNA encoding a SARS-CoV-2 Spikeprotein or an immunogenic fragment thereof from the Wuhan strain. Insome embodiments, BD₃ comprises an RNA encoding a Spike protein or animmunogenic fragment thereof from a SARS-CoV-2 strain that is prevalentand/or spreading rapidly in a relevant jurisdiction. In someembodiments, BD₃ comprises an RNA encoding a SARS-CoV-2 Spike protein oran immunogenic fragment thereof comprising one or more mutations from analpha variant. In some embodiments, BD₃ comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof comprisingone or more mutations from a delta variant. In some embodiments, BD₃comprises an RNA encoding a SARS-CoV-2 Spike protein or an immunogenicfragment thereof comprising one or more mutations from a beta variant.In some embodiments, BD₃ comprises an RNA encoding a SARS-CoV-2 Spikeprotein or an immunogenic fragment thereof comprising one or moremutations from an Omicron variant (e.g., a BA.4/5, BA.1, BA.2, XBB,XBB.1, or BQ.1 Omicron variant).

In some embodiments, BD₃ comprises an RNA encoding a SARS-CoV-2 Spikeprotein or an immunogenic fragment thereof from the Wuhan strain and oneor more RNA encoding a Spike protein or an immunogenic fragment thereoffrom a SARS-CoV-2 strain that is prevalent and/or spreading rapidly in arelevant jurisdiction. In some embodiments, BD₃ comprises an RNAencoding a SARS-CoV-2 Spike protein or an immunogenic fragment thereoffrom the Wuhan strain and an RNA encoding a SARS-CoV-2 Spike protein oran immunogenic fragment thereof comprising one or more mutations from aalpha variant. In some embodiments, BD₃ comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from adelta variant. In some embodiments, BD₃ comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from abeta variant. In some embodiments, BD₃ comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from anOmicron variant (e.g., a BA.4/5, BA.1, BA.2, XBB, XBB.1, or BQ.1 Omicronvariant).

In some embodiments, BD_(n) comprises an RNA encoding a SARS-CoV-2 Spikeprotein or an immunogenic fragment thereof from the Wuhan strain. Insome embodiments, BD_(n) comprises an RNA encoding a Spike protein or animmunogenic fragment thereof from a SARS-CoV-2 strain that is prevalentand/or spreading rapidly in a relevant jurisdiction. In someembodiments, BD_(n) comprises an RNA encoding a SARS-CoV-2 Spike proteinor an immunogenic fragment thereof comprising one or more mutations froman alpha variant. In some embodiments, BD_(n) comprises an RNA encodinga SARS-CoV-2 Spike protein or an immunogenic fragment thereof comprisingone or more mutations from a delta variant. In some embodiments, BD_(n)comprises an RNA encoding a SARS-CoV-2 Spike protein or an immunogenicfragment thereof comprising one or more mutations from a beta variant.In some embodiments, BD_(n) comprises an RNA encoding a SARS-CoV-2 Spikeprotein or an immunogenic fragment thereof comprising one or moremutations from an Omicron variant (e.g., a BA.4/5, BA.1, BA.2, XBB,XBB.1, or BQ.1 Omicron variant).

In some embodiments, BD_(n) comprises an RNA encoding a SARS-CoV-2 Spikeprotein or an immunogenic fragment thereof from the Wuhan strain and oneor more RNA encoding a Spike protein or an immunogenic fragment thereoffrom a SARS-CoV-2 strain that is prevalent and/or spreading rapidly in arelevant jurisdiction. In some embodiments, BD_(n) comprises an RNAencoding a SARS-CoV-2 Spike protein or an immunogenic fragment thereoffrom the Wuhan strain and an RNA encoding a SARS-CoV-2 Spike protein oran immunogenic fragment thereof comprising one or more mutations from aalpha variant. In some embodiments, BD_(n) comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from adelta variant. In some embodiments, BD_(n) comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from abeta variant. In some embodiments, BD_(n) comprises an RNA encoding aSARS-CoV-2 Spike protein or an immunogenic fragment thereof from theWuhan strain and an RNA encoding a SARS-CoV-2 Spike protein or animmunogenic fragment thereof comprising one or more mutations from anOmicron variant (e.g., a BA.4/5, BA.1, BA.2, XBB, XBB.1, or BQ.1 Omicronvariant).

In some embodiments, BD₁, BD₂, BD₃, and BD_(n) can each independentlycomprise a plurality of (e.g., at least two) mRNA compositions describedherein. In some embodiments BD₁, BD₂, BD₃, and BD_(n) can eachindependently comprise a first and a second mRNA composition. In someembodiments, BD₁, BD₂, BD₃, and BD_(n) can each independently comprise aplurality of (e.g., at least two) mRNA compositions, wherein, at leastone of the plurality of mRNA compositions comprises BNT162b2 (e.g., asdescribed herein). In some embodiments, at least one of a plurality ofmRNA compositions comprises an mRNA encoding a SARS-CoV-2 S protein oran immunogenic fragment thereof from a different SARS-CoV-2 variant(e.g., a variant that is prevalent or rapidly spreading in a relevantjurisdiction, e.g., a variant disclosed herein). In some embodiments, atleast one of a plurality of mRNA compositions comprises an mRNA encodinga SARS-CoV-2 S protein or an immunogenic fragment thereof from a Wuhanstrain of SARS-CoV-2. In some embodiments, at least one of a pluralityof mRNA compositions comprises an RNA encoding a SARS-CoV-2 S protein oran immunogenic fragment thereof comprising one or more mutations from avariant that is prevalent and/or spreading rapidly in a relevantjurisdiction. In some embodiments, at least one of a plurality of mRNAcompositions comprises an mRNA encoding a SARS-CoV-2 S protein or animmunogenic fragment thereof comprising one or more mutations from analpha variant. In some embodiments, at least one of a plurality of mRNAcompositions comprises an mRNA encoding a SARS-CoV-2 S protein or animmunogenic fragment thereof comprising one or more mutations from adelta variant. In some embodiments, at least one of a plurality of mRNAcompositions comprises an mRNA encoding a SARS-CoV-2 S protein or animmunogenic fragment thereof comprising one or more mutations from anOmicron variant (e.g., a BA.4/5, BA.1, BA.2, XBB, XBB.1, or BQ.1 Omicronvariant).

In some embodiments, a plurality of mRNA compositions given in BD₁, BD₂,BD₃, and/or BD_(n) can each independently comprise at least twodifferent mRNA constructs (e.g., mRNA constructs having differingprotein-encoding sequences). For example, in some embodiments aplurality of mRNA compositions given in BD₁, BD₂, BD₃, and/or BD₁ caneach independently comprise an mRNA encoding a SARS-CoV-2 S protein oran immunogenic fragment thereof from a Wuhan strain of SARS-CoV-2 and anmRNA encoding a SARS-CoV-2 S protein or an immunogenic fragment thereofcomprising one or more mutations from a variant that is prevalent and/orspreading rapidly in a relevant jurisdiction. In some embodiments aplurality of mRNA compositions given in BD₁, BD₂, BD₃, and/or BD_(n) caneach independently comprise an mRNA encoding a SARS-CoV-2 S protein oran immunogenic fragment thereof derived from a Wuhan strain ofSARS-CoV-2 and an mRNA encoding a SARS-CoV-2 S protein or an immunogenicfragment thereof comprising one or more mutations from a variant that isprevalent and/or spreading rapidly in a relevant jurisdiction. In somesuch embodiments, a variant can be an alpha variant. In some suchembodiments, a variant can be a delta variant. In some such embodimentsa variant can be an Omicron variant (e.g., a BA.4/5, BA.1, BA.2, XBB,XBB.1, or BQ.1 Omicron variant).

In some embodiments, a plurality of mRNA compositions given in BD₁, BD₂,BD₃, and/or BD_(n) can each independently comprise at least two mRNAseach encoding a SARS-CoV-2 S protein or an immunogenic fragment thereofcomprising one or more mutations from a distinct variant that isprevalent and/or spreading rapidly in a relevant jurisdiction. In someembodiments a plurality of mRNA compositions given in BD₁, BD₂, BD₃,and/or BD_(n) can each independently comprise an mRNA encoding aSARS-CoV-2 S protein or an immunogenic fragment thereof from an alphavariant and an mRNA encoding a SARS-CoV-2 S protein or an immunogenicfragment thereof comprising one or more mutations from a delta variant.In some embodiments a plurality of mRNA compositions given in BD₁, BD₂,BD₃, and/or BD_(n) can each independently comprise an mRNA encoding aSARS-CoV-2 S protein or an immunogenic fragment thereof from an alphavariant and an mRNA encoding a SARS-CoV-2 S protein or an immunogenicfragment thereof comprising one or more mutations from an Omicronvariant (e.g., a BA.4/5, BA.1, BA.2, XBB, XBB.1, or BQ.1 Omicronvariant). In some embodiments a plurality of mRNA compositions given inBD₁, BD₂, BD₃, and/or BD_(n) can each independently comprise an mRNAencoding a SARS-CoV-2 S protein or an immunogenic fragment thereof froma delta variant and an mRNA encoding a SARS-CoV-2 S protein or animmunogenic fragment thereof comprising one or more mutations from anOmicron variant (e.g., a BA.4/5, BA.1, BA.2, XBB, XBB.1, or BQ.1 Omicronvariant).

In some embodiments, a plurality of mRNA compositions given in BD₁, BD₂,BD₃, and/or BD_(n) are separately administered to a subject, forexample, in some embodiments via intramuscular injection, at differentinjection sites. For example, in some embodiments, a first and secondmRNA composition given in BD₁, BD₂, BD₃, and/or BD_(n) are separatelyadministered to different arms of a subject via intramuscular injection.

In some embodiments, the length of time between BD₁ and BD₂ (BI₁) is atleast about 1 week, at least about 2 weeks, at least about 3 weeks, orat least about 4 weeks. In some embodiments, BI₁ is about 1 week toabout 12 weeks. In some embodiments, BI₁ is about 1 week to about 10weeks. In some embodiments, BI₁ is about 2 weeks to about 10 weeks. Insome embodiments, BI₁ is about 2 weeks to about 8 weeks. In someembodiments, BI₁ is about 3 weeks to about 8 weeks. In some embodiments,BI₁ is about 4 weeks to about 8 weeks. In some embodiments, BI₁ is about6 weeks to about 8 weeks. In some embodiments BI₁ is about 3 to about 4weeks. In some embodiments, BI₁ is about 1 week. In some embodiments,BI₁ is about 2 weeks. In some embodiments, BI₁ is about 3 weeks. In someembodiments, BI₁ is about 4 weeks. In some embodiments, BI₁ is about 5weeks. In some embodiments, BI₁ is about 6 weeks. In some embodiments,BI₁ is about 7 weeks. In some embodiments, BI₁ is about 8 weeks. In someembodiments, BI₁ is about 9 weeks. In some embodiments, BI₁ is about 10weeks.

In some embodiments, the length of time between BD₂ and BD₃ (BI₂) is atleast about 1 week, at least about 2 weeks, or at least about 3 weeks.In some embodiments, BI₂ is about 1 week to about 12 weeks. In someembodiments, BI₂ is about 1 week to about 10 weeks. In some embodiments,BI₂ is about 2 weeks to about 10 weeks. In some embodiments, BI₂ isabout 2 weeks to about 8 weeks. In some embodiments, BI₂ is about 3weeks to about 8 weeks. In some embodiments, BI₂ is about 4 weeks toabout 8 weeks. In some embodiments, BI₂ is about 6 weeks to about 8weeks. In some embodiments BI₂ is about 3 to about 4 weeks. In someembodiments, BI₂ is about 1 week. In some embodiments, BI₂ is about 2weeks. In some embodiments, BI₂ is about 3 weeks. In some embodiments,BI₂ is about 4 weeks. In some embodiments, BI₂ is about 5 weeks. In someembodiments, BI₂ is about 6 weeks. In some embodiments, BI₂ is about 7weeks. In some embodiments, BI₂ is about 8 weeks. In some embodiments,BI₂ is about 9 weeks. In some embodiments, BI₂ is about 10 weeks.

In some embodiments, the length of time between BD₃ and a subsequentdose that is part of the Booster Dosing Regimen, or between doses forany dose beyond BD₃ (BI_(n)) is each separately and independentlyselected from: about 1 week or more, about 2 weeks or more, or about 3weeks or more. In some embodiments, BI_(n) is about 1 week to about 12weeks. In some embodiments, BI_(n) is about 1 week to about 10 weeks. Insome embodiments, BI_(n) is about 2 weeks to about 10 weeks. In someembodiments, BI_(n) is about 2 weeks to about 8 weeks. In someembodiments, BI_(n) is about 3 weeks to about 8 weeks. In someembodiments, BI_(n) is about 4 weeks to about 8 weeks. In someembodiments, BI_(n) is about 6 weeks to about 8 weeks. In someembodiments BI_(n) is about 3 to about 4 weeks. In some embodiments,BI_(n) is about 1 week. In some embodiments, BI_(n) is about 2 weeks. Insome embodiments, BI_(n) is about 3 weeks. In some embodiments, BI_(n)is about 4 weeks. In some embodiments, BI_(n) is about 5 weeks. In someembodiments, BI_(n) is about 6 weeks. In some embodiments, BI_(n) isabout 7 weeks. In some embodiments, BI_(n) is about 8 weeks. In someembodiments, BI_(n) is about 9 weeks. In some embodiments, BI_(n) isabout 10 weeks.

In some embodiments, one or more compositions adminstered in BD₁ areformulated in a Tris buffer. In some embodiments, one or morecompositions administered in BD₂ are formulated in a Tris buffer. Insome embodiments, one or more compositions administering in BD₃ areformulated in a Tris buffer. In some embodiments, one or morecompositions administered in BD₃ are formulated in a Tris buffer.

In some embodiments, the Booster dosing regimen comprises administeringtwo or more mRNA compositions described herein, and at least two of themRNA compositions have different formulations. In some embodiments, theBooster dosing regimen comprises BD₁ and BD₂, where BD₁ comprisesadministering an mRNA formulated in a Tris buffer and BD₂ comprisesadministering an mRNA formulated in a PBS buffer. In some embodiments,the Booster dosing regimen comprises BD₁ and BD₂, where BD₁ comprisesadministering an mRNA formulated in a PBS buffer and BD₂ comprisesadministering an mRNA formulated in a Tris buffer.

In some embodiments, one or more mRNA compositions given in BD₁, BD₂,BD₃, and/or BD_(n) can be administered in combination with anothervaccine. In some embodiments, another vaccine is for a disease that isnot COVID-19. In some embodiments, the disease is one that increasesdeleterious effects of SARS-CoV-2 when a subject is coinfected with thedisease and SARS-CoV-2. In some embodiments, the disease is one thatincreases the transmission rate of SARS-CoV-2 when a subject iscoinfected with the disease and SARS-CoV-2. In some embodiments, anothervaccine is a different commerically available vaccine. In someembodiments, the different commercially available vaccine is an RNAvaccine. In some embodiments, the different commercially availablevaccine is a polypeptide-based vaccine. In some embodiments, anothervaccine (e.g., as described herein) and one or more mRNA compositionsgiven in BD₁, BD₂, BD₃, and/or BD_(n) are separately administered, forexample, in some embodiments via intramuscular injection, at differentinjection sites. For example, in some embodiments, an influenza vaccineand one or more SARS-CoV-2 mRNA compositions described herein given inBD₁, BD₂, BD₃, and/or BD_(n) are separately administered to differentarms of a subject via intramuscular injection.

Additional Booster Regimens

In some embodiments, methods of vaccination disclosed herein compriseadministering more than one Booster Dosing Regimen. In some embodiments,more than one Booster Dosing Regimen may need to be administered toincrease neutralizing antibody response. In some embodiments, more thanone booster dosing regimen may be needed to counteract a SARS-CoV-2strain that has been shown to have a high likelihood of evading immuneresponse elicited by vaccines that a patient has previously received. Insome embodiments, an additional Booster Dosing Regimen is administeredto a patient who has been determined to produce low concentrations ofneutralizing antibodies. In some embodiments, an additional boosterdosing regimen is administered to a patient who has been determined tohave a high likelihood of being susceptible to SARS-CoV-2 infection,despite previous vaccination (e.g., an immunocompromised patient, acancer patient, and/or an organ transplant patient).

The description provided above for the first Booster Dosing Regimen alsodescribes the one or more additional Booster Dosing Regimens. Theinterval of time between the first Booster Dosing Regimen and a secondBooster Dosing Regimen, or between subsequent Booster Dosing Regimenscan be any of the acceptable intervals of time described above betweenthe Primary Dosing Regimen and the First Booster Dosing Regimen.

In some embodiments, a dosing regimen comprises a primary regimen and abooster regimen, wherein at least one dose given in the primary regimenand/or the booster regimen comprises a composition comprising an RNAthat encodes a S protein or immunogenic fragment thereof from a variantthat is prevalent or is spreading rapidly in a relevant jurisdiction(e.g., Omicron variant as described herein). For example, in someembodiments, a primary regimen comprises at least 2 doses of BNT162b2(e.g., encoding a Wuhan strain), for example, given at least 3 weeksapart, and a booster regimen comprises at least 1 dose of a compositioncomprising RNA that encodes a S protein or immunogenic fragment thereoffrom a variant that is prevalent or is spreading rapidly in a relevantjurisdiction (e.g., Omicron variant as described herein). In some suchembodiments, such a dose of a booster regimen may further comprise anRNA that encodes a S protein or immunogenic fragment thereof from aWuhan strain, which can be administered with an RNA that encodes a Sprotein or immunogenic fragment thereof from a variant that is prevalentor is spreading rapidly in a relevant jurisdiction (e.g., Omicronvariant as described herein), as a single mixture, or as two separatecompositions, for example, in 1:1 weight ratio. In some embodiments, abooster regimen can also comprise at least 1 dose of BNT162b2, which canbe administered as a first booster dose or a subsequent booster dose.

In some embodiments, an RNA composition described herein is given as abooster at a dose that is higher than the doses given during a primaryregimen (primary doses) and/or the dose given for a first booster, ifany. For example, in some embodiments, such a dose may be 60 ug; or insome embodiments such a dose may be higher than 30 ug and lower than 60ug (e.g., 55 ug, 50 ug, or lower). In some embodiments, an RNAcomposition described herein is given as a booster at least 3-12 monthsor 4-12 months, or 5-12 months, or 6-12 months after the last dose(e.g., the last dose of a primary regimen or a first dose of a boosterregimen). In some embodiments, the primary doses and/or the firstbooster dose (if any) may comprise BNT162b2, for example at 30 ug perdose.

In some embodiments, an RNA composition described herein comprises anRNA encoding a polypeptide as set forth in SEQ ID NO: 49 or animmunogenic fragment thereof, or a variant thereof (e.g., having atleast 70% or more, including, e.g., at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or higher,identity to SEQ ID NO: 49). In some embodiments, an RNA compositioncomprises an RNA that includes the sequence of SEQ ID NO: 50 or avariant thereof (e.g., having at least 70% or more, including, e.g., atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or higher, identity to SEQ ID NO: 50). In someembodiments, an RNA composition comprises an RNA that includes thesequence of SEQ ID NO: 51 or a variant thereof (e.g., having at least70% or more, including, e.g., at least 80%, at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or higher,identity to SEQ ID NO: 51).

In some embodiments, an RNA composition described herein comprises anRNA encoding a polypeptide as set forth in SEQ ID NO: 55 or animmunogenic fragment thereof, or a variant thereof (e.g., having atleast 70% or more, including, e.g., at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or higher,identity to SEQ ID NO: 55. In some embodiments, an RNA compositioncomprises an RNA that includes the sequence of SEQ ID NO: 56 or avariant thereof (e.g., having at least 70% or more, including, e.g., atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or higher, identity to SEQ ID NO: 56). In someembodiments, an RNA composition comprises an RNA that includes thesequence of SEQ ID NO: 57 or a variant thereof (e.g., having at least70% or more, including, e.g., at least 80%, at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or higher,identity to SEQ ID NO: 57).

In some embodiments, an RNA composition described herein comprises anRNA encoding a polypeptide as set forth in SEQ ID NO: 58 or animmunogenic fragment thereof, or a variant thereof (e.g., having atleast 70% or more, including, e.g., at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or higher,identity to SEQ ID NO: 58). In some embodiments, an RNA compositioncomprises an RNA that includes the sequence of SEQ ID NO: 59 or avariant thereof (e.g., having at least 70% or more, including, e.g., atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or higher, identity to SEQ ID NO: 59). In someembodiments, an RNA composition comprises an RNA that includes thesequence of SEQ ID NO: 60 or a variant thereof (e.g., having at least70% or more, including, e.g., at least 80%, at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or higher,identity to SEQ ID NO: 60).

In some embodiments, an RNA composition described herein comprises anRNA encoding a polypeptide as set forth in SEQ ID NO: 61 or animmunogenic fragment thereof, or a variant thereof (e.g., having atleast 70% or more, including, e.g., at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or higher,identity to SEQ ID NO: 61). In some embodiments, an RNA compositioncomprises an RNA that includes the sequence of SEQ ID NO: 62a or avariant thereof (e.g., having at least 70% or more, including, e.g., atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or higher, identity to SEQ ID NO: 62a). In someembodiments, an RNA composition comprises an RNA that includes thesequence of SEQ ID NO: 63a or a variant thereof (e.g., having at least70% or more, including, e.g., at least 80%, at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or higher,identity to SEQ ID NO: 63a).

In some embodiments, the formulations disclosed herein can be used tocarry out any of the dosing regimens described in Table 19 (below).

TABLE 19 Exemplary Dosing Regimens: Primary Regimen Time between theBooster Regimen Time last dose of a Time Between Dose 1 and Primaryregimen Between Dose 1 and Dose 1 Dose 2 Doses 1 Dose 2 and a first doseof Dose 1 Dose 2 Doses 1 Dose 2 # (μg RNA) (μg RNA) and 2 FormulationBooster Regimen (μg RNA) (μg RNA) and 2 Formulation 1 30 30 2 to 8 weeksPBS At least 2 months 30 N/A¹ N/A PBS 2 30 30 2 to 8 weeks PBS At least3 months 30 N/A¹ N/A PBS 3 30 30 2 to 8 weeks PBS 6 to 12 months 30 N/A¹N/A PBS 4 30 30 2 to 8 weeks PBS or Tris 4 to 12 months 15 N/A¹ N/A PBSor Tris 5 30 30 2 to 8 weeks PBS or Tris 4 to 12 months 10 N/A¹ N/A PBSor Tris 6 30 30 2 to 8 weeks PBS or Tris 4 to 12 months 30 30 4 to 12PBS or Tris months 7 30 30 2 to 8 weeks PBS or Tris 4 to 12 months 30 154 to 12 PBS or Tris months 8 30 30 2 to 8 weeks PBS or Tris 4 to 12months 30 10 4 to 12 PBS or Tris months 9 30 30 2 to 8 weeks PBS or Tris4 to 12 months 30 60 4 to 12 PBS or Tris months 10 30 30 2 to 8 weeksPBS or Tris 4 to 12 months 30 >30 to <60 4 to 12 PBS or Tris months 1130 30 2 to 8 weeks PBS or Tris 4 to 12 months 30 50 4 to 12 PBS or Trismonths 12 30 30 2 to 8 weeks PBS At least 6 months 30 N/A¹ N/A PBS 13 3030 ~21 days PBS At least 2 months 30 N/A¹ N/A PBS 14 30 30 ~21 days PBSAt least 3 months 30 N/A¹ N/A PBS 15 30 30 ~21 days PBS 6 to 12 months30 N/A¹ N/A PBS 16 30 30 ~21 days PBS At least 6 months 30 N/A¹ N/A PBS17 30 30 21 days PBS At least 6 months 15 15 ~21 days PBS 18 30 30 21days PBS At least 6 months 15 15 ~21 days PBS 19 30 30 2 to 8 weeks PBSAt least 2 months 30 N/A¹ N/A Tris 20 30 30 2 to 8 weeks PBS At least 3months 30 N/A¹ N/A Tris 21 30 30 2 to 8 weeks PBS 6 to 12 months 30 N/A¹N/A Tris 22 30 30 2 to 8 weeks PBS At least 6 months 30 N/A¹ N/A Tris 2330 30 ~21 days PBS At least 2 months 30 N/A¹ N/A Tris 24 30 30 ~21 daysPBS At least 3 months 30 N/A¹ N/A Tris 25 30 30 ~21 days PBS 6 to 12months 30 N/A¹ N/A Tris 26 30 30 ~21 days PBS At least 6 months 30 N/A¹N/A Tris 27 30 30 21 days PBS At least 6 months 15 15 ~21 days Tris 2830 30 21 days PBS At least 6 months 15 15 ~21 days Tris 29 30 30 2 to 8weeks Tris At least 2 months 30 N/A¹ N/A Tris 30 30 30 2 to 8 weeks TrisAt least 3 months 30 N/A¹ N/A Tris 31 30 30 2 to 8 weeks Tris 6 to 12months 30 N/A¹ N/A Tris 32 30 30 2 to 8 weeks Tris At least 6 months 30N/A¹ N/A Tris 33 30 30 ~21 days Tris At least 2 months 30 N/A¹ N/A Tris34 30 30 ~21 days Tris At least 3 months 30 N/A¹ N/A Tris 35 30 30 ~21days Tris 6 to 12 months 30 N/A¹ N/Å Tris 36 30 30 ~21 days Tris Atleast 6 months 30 N/A¹ N/A Tris 37 30 30 21 days Tris At least 6 months15 15 ~21 days Tris 38 30 30 21 days Tris At least 6 months 15 15 ~21days Tris 39 10 10 2 to 8 weeks Tris At least 2 months 10 N/A¹ N/A Tris40 10 10 2 to 8 weeks Tris At least 3 months 10 N/A¹ N/A Tris 41 10 10 2to 8 weeks Tris 6 to 12 months 10 N/A¹ N/A Tris 42 10 10 2 to 8 weeksTris At least 6 months 10 N/A¹ N/A Tris 43 10 10 ~21 days Tris At least2 months 10 N/A¹ N/A Tris 44 10 10 ~21 days Tris At least 3 months 10N/A¹ N/A Tris 45 10 10 ~21 days Tris 6 to 12 months 10 N/A¹ N/A Tris 4610 10 ~21 days Tris At least 6 months 10 N/A¹ N/A Tris 47 3 3 2 to 8weeks Tris At least 2 months 3 N/A¹ N/A Tris 48 3 3 2 to 8 weeks Tris Atleast 3 months 3 N/A¹ N/A Tris 49 3 3 2 to 8 weeks Tris 6 to 12 months 3N/A¹ N/A Tris 50 3 3 2 to 8 weeks Tris At least 6 months 3 N/A¹ N/A Tris51 3 3 ~21 days Tris At least 2 months 3 N/A¹ N/A Tris 52 3 3 ~21 daysTris At least 3 months 3 N/A¹ N/A Tris 53 3 3 ~21 days Tris 6 to 12months 3 N/A¹ N/A Tris 54 3 3 ~21 days Tris At least 6 months 3 N/A¹ N/ATris ¹N/A refers to no dose necessary.

In some embodiments of certain exemplary dosing regimens as described inTable 19 above, an RNA composition described herein (e.g., comprisingRNA encoding a variant described herein) is given in a first dose of aprimary regimen. In some embodiments of certain exemplary dosingregimens as described in Table 19 above, an RNA composition describedherein (e.g., comprising RNA encoding a variant described herein) isgiven in a second dose of a primary regimen. In some embodiments ofcertain exemplary dosing regimens as described in Table 19 above, an RNAcomposition described herein (e.g., comprising RNA encoding a variantdescribed herein) is given in a first dose and a second dose of aprimary regimen. In some embodiments of certain exemplary dosingregimens as described in Table 19 above, an RNA composition describedherein (e.g., comprising RNA encoding a variant described herein) isgiven in a first dose of a booster regimen. In some embodiments ofcertain exemplary dosing regimens as described in Table 19 above, an RNAcomposition described herein (e.g., comprising RNA encoding a variantdescribed herein) is given in a second dose of a booster regimen. Insome embodiments of certain exemplary dosing regimens as described inTable 19 above, an RNA composition described herein (e.g., comprisingRNA encoding a variant described herein) is given in a first dose and asecond dose of a booster regimen. In some embodiments of certainexemplary dosing regimens as described in Table 19 above, an RNAcomposition described herein (e.g., comprising RNA encoding a variantdescribed herein) is given in a first dose and a second dose of aprimary regimen and also in at least one dose of a booster regimen. Insome embodiments of certain exemplary dosing regimens as described inTable 19 above, an RNA composition described herein (e.g., comprisingRNA encoding a variant described herein) is given in at least one dose(including, e.g., at least two doses) of a booster regimen and BNT162b2is given in a primary regimen. In some embodiments of certain exemplarydosing regimens as described in Table 19 above, an RNA compositiondescribed herein (e.g., comprising RNA encoding a variant describedherein) is given in a second dose of a booster regimen and BNT162b2 isgiven in a primary regimen and in a first dose of a booster regimen. Insome embodiments, an RNA composition described herein (e.g., comprisingRNA encoding a variant described herein) comprises an RNA encoding apolypeptide as set forth in SEQ ID NO: 49 or an immunogenic fragmentthereof, or a variant thereof (e.g., having at least 70% or more,including, e.g., at least 80%, at least 85%, at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, or higher, identity to SEQ IDNO: 49). In some embodiments, an RNA composition described herein (e.g.,comprising RNA encoding a variant described herein) comprises an RNAthat includes the sequence of SEQ ID NO: 50 or a variant thereof (e.g.,having at least 70% or more, including, e.g., at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, or higher, identity to SEQ ID NO: 50). In some embodiments, an RNAcomposition described herein (e.g., comprising RNA encoding a variantdescribed herein) comprises an RNA that includes the sequence of SEQ IDNO: 51 or a variant thereof (e.g., having at least 70% or more,including, e.g., at least 80%, at least 85%, at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, or higher, identity to SEQ IDNO: 51).

In some embodiments, an RNA composition described herein comprises anRNA encoding a polypeptide as set forth in SEQ ID NO: 55 or animmunogenic fragment thereof, or a variant thereof (e.g., having atleast 70% or more, including, e.g., at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or higher,identity to SEQ ID NO: 55). In some embodiments, an RNA compositioncomprises an RNA that includes the sequence of SEQ ID NO: 56 or avariant thereof (e.g., having at least 70% or more, including, e.g., atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or higher, identity to SEQ ID NO: 56). In someembodiments, an RNA composition comprises an RNA that includes thesequence of SEQ ID NO: 57 or a variant thereof (e.g., having at least70% or more, including, e.g., at least 80%, at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or higher,identity to SEQ ID NO: 57).

In some embodiments, an RNA composition described herein comprises anRNA encoding a polypeptide as set forth in SEQ ID NO: 58 or animmunogenic fragment thereof, or a variant thereof (e.g., having atleast 70% or more, including, e.g., at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or higher,identity to SEQ ID NO: 58). In some embodiments, an RNA compositioncomprises an RNA that includes the sequence of SEQ ID NO: 59 or avariant thereof (e.g., having at least 70% or more, including, e.g., atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or higher, identity to SEQ ID NO: 59). In someembodiments, an RNA composition comprises an RNA that includes thesequence of SEQ ID NO: 60 or a variant thereof (e.g., having at least70% or more, including, e.g., at least 80%, at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or higher,identity to SEQ ID NO: 60).

In some embodiments, an RNA composition described herein comprises anRNA encoding a polypeptide as set forth in SEQ ID NO: 61 or animmunogenic fragment thereof, or a variant thereof (e.g., having atleast 70% or more, including, e.g., at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or higher,identity to SEQ ID NO: 61). In some embodiments, an RNA compositioncomprises an RNA that includes the sequence of SEQ ID NO: 62a or avariant thereof (e.g., having at least 70% or more, including, e.g., atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or higher, identity to SEQ ID NO: 62a). In someembodiments, an RNA composition comprises an RNA that includes thesequence of SEQ ID NO: 63a or a variant thereof (e.g., having at least70% or more, including, e.g., at least 80%, at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or higher,identity to SEQ ID NO: 63a).

In some embodiments, such an RNA composition described herein (e.g.,comprising RNA encoding a variant described herein) can further compriseRNA encoding a S protein or an immunogenic fragment thereof of adifferent strain (e.g., a Wuhan strain). By way of example, in someembodiments, a second dose of a booster regimen of Regimens #9-11 asdescribed in Table 19 above can comprise an RNA composition describedherein (e.g., comprising RNA encoding a variant described herein such asOmicron, for example, in one embodiment RNA as described in thisExample) and a BNT162b2 construct, for example, in 1:1 weight ratio.

In some embodiments of Regimen #6 as described in Table 19 above, afirst dose and a second dose of a primary regimen and a first dose and asecond dose of a booster regimen each comprise an RNA compositiondescribed herein (e.g., comprising RNA encoding a variant describedherein such as Omicron, for example, in one embodiment RNA as describedin this Example). In some such embodiments, a second dose of a boosterregimen may not be necessary.

In some embodiments of Regimen #6 as described in Table 19 above, afirst dose and a second dose of a primary regimen and a first dose and asecond dose of a booster regimen each comprise an RNA compositiondescribed herein (e.g., comprising RNA encoding a variant describedherein such as Omicron, for example, in one embodiment RNA as describedin this Example). In some such embodiments, a second dose of a boosterregimen may not be necessary.

In some embodiments of Regimen #6 as described in Table 19 above, afirst dose and a second dose of a primary regimen each comprise aBNT162b2 construct, and a first dose and a second dose of a boosterregimen each comprise an RNA composition described herein (e.g.,comprising RNA encoding a variant described herein such as Omicron, forexample, in one embodiment RNA as described in this Example). In somesuch embodiments, a second dose of a booster regimen may not benecessary.

In some embodiments of Regimen #6 as described in Table 19 above, afirst dose and a second dose of a primary regimen and a first dose of abooster regimen each comprise a BNT162b2 construct, and a second dose ofa booster regimen comprises an RNA composition described herein (e.g.,comprising RNA encoding a variant described herein such as Omicron, forexample, in one embodiment RNA as described in this Example).

Example 7: Omicron BA.1 Breakthrough Infection Drives Cross-VariantNeutralization and Memory B Cell Formation

The present Example shows that an Omicron BA.1 breakthrough infection inindividuals double- and triple-vaccinated with BNT162b2 drives crossvariant neutralization and memory B cell formation, including productionof neutralizing antibodies and B cell responses toward an Omicron BA.1variant. One of ordinary skill in the art reading the present Examplewill understand that such findings can be extended to administration ofan mRNA vaccine comprising an RNA encoding a SARS-CoV-2 S protein havingmutations characteristic of an Omicron BA.1 variant (e.g., ones asdescribed herein) to subjects who were previously administered two orthree doses of SARS-CoV-2 vaccines (e.g., in some embodiments developedbased on a S protein from a Wuhan-Hu-1 strain).

Omicron is the evolutionarily most distinct SARS-CoV-2 variant ofconcern (VOC) to date. To address how Omicron breakthrough infection canpotentially reshape SARS-CoV-2 recognition in vaccinated individuals,the effects of Omicron BA.1 breakthrough infection were investigated onserum neutralization and B_(MEM) cell antigen recognition in BNT162b2double- and triple-vaccinated individuals. Omicron BA.1 breakthroughinfection induced broad neutralization of VOCs including Omicron BA.1,with substantially stronger neutralization compared to OmicronBA.1-naïve double- and triple-vaccines. Broad recognition of VOCs byB_(MEM) cells from BNT162b2 double- and triple-vaccinated individualswas boosted by Omicron BA.1 breakthrough infection, with recognitionprimarily against conserved epitopes shared broadly between variantsrather than Omicron BA.1-specific epitopes. The data presented hereindemonstrate that an Omicron BA.1 breakthrough infection efficientlybroadens neutralizing antibody and/or B cell responses towards multiplevariants and suggest that a vaccine adapted to the Omicron BA.1 Sprotein may be able to reshape the immune repertoire.

Introduction

Containment of the current COVID-19 pandemic requires the generation ofdurable and sufficiently broad immunity that provides protection againstcirculating and future variants of SARS-CoV-2. The titer of neutralizingantibodies to SARS-CoV-2, and the binding of antibodies to the spike (S)glycoprotein and its receptor-binding domain (RBD) are consideredcorrelates of protection against infection (D. S. Khoury et al.,“Neutralizing antibody levels are highly predictive of immune protectionfrom symptomatic SARS-CoV-2 infection,” Nature medicine. 27, 1205-1211(2021), doi:10.1038/s41591-021-01377-8; and P. B. Gilbert et al.,“Immune correlates analysis of the mRNA-1273 COVID-19 vaccine efficacyclinical trial,” Science (New York, N.Y.). 375, 43-50 (2022),doi:10.1126/science.abm3425). Currently available vaccines are based onthe ancestral Wuhan-Hu-1 strain and induce antibodies with aneutralizing capacity that exceeds the breadth elicited by infectionwith the Wuhan strain, or with variants of concern (VOCs) (K. Röltgen etal., “Immune imprinting, breadth of variant recognition, and germinalcenter response in human SARS-CoV-2 infection and vaccination,” Cell(2022), doi:10.1016/j.cell.2022.01.018). However, protective titers waneover time (J. P. Evans et al., “Neutralizing antibody responses elicitedby SARS-CoV-2 mRNA vaccination wane over time and are boosted bybreakthrough infection,” Science translational medicine, eabn8057(2022), doi:10.1126/scitranslmed.abn8057; S. Yamayoshi et al., “Antibodytiters against SARS-CoV-2 decline, but do not disappear for severalmonths,” Eclinical Medicine, 32, 100734 (2021),doi:10.1016/j.eclinm.2021.100734; W. N. Chia et al., “Dynamics ofSARS-CoV-2 neutralising antibody responses and duration of immunity,”The Lancet Microbe, 2, e240-e249 (2021),doi:10.1016/S2666-5247(21)00025-2; Y. Goldberg et al., “Waning Immunityafter the BNT162b2 Vaccine in Israel,” The New England journal ofmedicine. 385, e85 (2021), doi:10.1056/NEJMoa2114228. and routinebooster vaccinations are thought to be needed to trigger recall immunityand maintain efficacy against new VOCs (A. R. Falsey et al., “SARS-CoV-2Neutralization with BNT162b2 Vaccine Dose 3,” The New England journal ofmedicine. 385, 1627-1629 (2021), doi:10.1056/NEJMc2113468; A. Choi etal., “Safety and immunogenicity of SARS-CoV-2 variant mRNA vaccineboosters in healthy adults,” Nature medicine. 27, 2025-2031 (2021),doi:10.1038/s41591-021-01527-y; and N. Andrews et al., “Effectiveness ofCOVID-19 booster vaccines against covid-19 related symptoms,hospitalisation and death in England,” Nature medicine (2022),doi:10.1038/s41591-022-01699-1.

Long-lived memory B (B_(MEM)) cells are the basis for the recallresponse upon antigen reencounter either by infection or boostervaccination. They play an important role in the maintenance andevolution of the antiviral antibody response against variants, sincelow-affinity selection mechanisms during the germinal center reactionand continued hypermutation of B_(MEM) cells expand the breadth of viralvariant recognition overtime (W. E. Purtha, et al., “Memory B cells, butnot long-lived plasma cells, possess antigen specificities for viralescape mutants,” The Journal of experimental medicine, 208, 2599-2606(2011) doi:10.1084/jem.20110740; and Y. Adachi et al., “Distinctgerminal center selection at local sites shapes memory B cell responseto viral escape,” The Journal of experimental medicine. 212, 1709-1723(2015), doi:10.1084/jem.20142284).

How vaccine-mediated protective immunity will evolve over time and willbe modified by iterations of exposure to COVID-19 vaccines andinfections with increasingly divergent viral variants, is of particularrelevance with the emergence of antigenically distinct VOCs. Omicron isthe evolutionarily most distant reported VOC with a hithertounprecedented number of amino acid alterations in its S glycoprotein,including at least 15 amino acid changes in the RBD and extensivechanges in the N-terminal domain (NTD). These alterations are predictedto affect most neutralizing antibody epitopes. In addition, Omicron ishighly transmissible, and its sublineages BA.1 and BA.2 have spreadrapidly across the globe, outcompeting Delta within weeks to become thedominant circulating VOC (W. Dejnirattisai et al., “SARS-CoV-2Omicron-B.1.1.529 leads to widespread escape from neutralizing antibodyresponses,” Cell. 185, 467-484.e15 (2022),doi:10.1016/j.cell.2021.12.046; and M. Hoffmann et al., “The Omicronvariant is highly resistant against antibody-mediated neutralization,”Cell. 185, 447-456.e11 (2022), doi:10.1016/j.cell.2021.12.032).

To date, over 1 billion people worldwide have been vaccinated with themRNA-based COVID-19 vaccine BNT162b2 and have received the primary2-dose series or further boosters. This vaccine is contributingsubstantially to the pattern of population immunity in many regions onwhich further immune editing and effects of currently spreading variantswill build upon.

To characterize the effect of Omicron BA.1 breakthrough infection on themagnitude and breadth of serum neutralizing activity and B_(MEM) cells,blood samples from individuals that were double- or triple-vaccinatedwith BNT162b2 were studied.

As understanding of the antigen-specific B cell memory pool is acritical determinant of an individual's ability to respond to newlyemerging variants, this data can help to guide vaccine development.

Results and Discussion

Cohorts and Sampling

Blood samples have been sourced from the biosample collection ofBNT162b2 vaccine trials, and a biobank of prospectively collectedsamples from vaccinated individuals with subsequent SARS-CoV-2 OmicronBA.1 breakthrough infection. Samples were selected to investigatebiomarkers in four independent groups, namely individuals who were (i)double- or (ii) triple-vaccinated with BNT162b2 without a prior orbreakthrough infection at the time of sample collection (BNT162b2²,BNT162b2³) and individuals who were (iii) double- or (iv)triple-vaccinated with BNT162b2 and who experienced breakthroughinfection with the SARS-CoV-2 Omicron BA.1 variant after a median ofapproximately 5 months or 4 weeks, respectively (BNT162b2²+Omi,BNT162b2³+Omi) (see materials and methods below). Immune sera were usedto characterize Omicron BA.1 infection-associated changes to themagnitude and the breadth of serum neutralizing activity. PBMCs wereused to characterize the VOC-specificity of peripheral B_(MEM) cellsrecognizing the respective full-length SARS-CoV-2 S protein or its RBD(FIG. 15 ).

Omicron Breakthrough Infection of BNT162b2 Double- and Triple-VaccinatedIndividuals Induces Broad Neutralization of Omicron BA.1, BA.2 and OtherVOCs

To evaluate the neutralizing activity of immune sera, two orthogonaltest systems were used: a well-characterized pseudovirus neutralizationtest (pVNT) to investigate the breadth of inhibition of virus entry in apropagation-deficient set-up, as well as a live SARS-CoV-2neutralization test (VNT) designed to evaluate neutralization duringmulticycle replication of authentic virus with the antibodies maintainedthroughout the entire test period. For the former, pseudoviruses bearingS proteins comprising mutations characteristic of Omicron sublineagesBA.1 or BA.2, other SARS-CoV-2 VOCs (Wuhan, Alpha, Beta, Delta) wereused to assess breadth while pseudoviruses bearing the S proteins ofSARS-CoV-1 (T. Li et al., “Phylogenetic supertree reveals detailedevolution of SARS-CoV-2,” Scientific reports, 10, 22366 (2020),doi:10.1038/s41598-020-79484-8) was used to detect potentialpan-Sarbecovirus neutralizing activity (C.-W. Tan et al.,“Pan-Sarbecovirus Neutralizing Antibodies in BNT162b2-ImmunizedSARS-CoV-1 Survivors,” The New England journal of medicine, 385,1401-1406 (2021), doi:10.1056/NEJMoa2108453).

As reported previously (A. R. Falsey et al., “SARS-CoV-2 Neutralizationwith BNT162b2 Vaccine Dose 3,” The New England journal of medicine, 385,1627-1629 (2021), doi:10.1056/NEJMc2113468; and C.-W. Tan et al.,“Pan-Sarbecovirus Neutralizing Antibodies in BNT162b2-ImmunizedSARS-CoV-1 Survivors,” The New England journal of medicine. 385,1401-1406 (2021), doi:10.1056/NEJMoa2108453), in Omicron-naïvedouble-vaccinated individuals 50% pseudovirus neutralization (pVN₅₀)geometric mean titers (GMTs) of Beta and Delta VOCs were reduced, andneutralization of both Omicron sublineages was virtually undetectable.In Omicron-naïve triple-vaccinated individuals, pVN₅₀ GMTs against alltested VOCs were substantially higher with robust neutralization ofAlpha, Beta and Delta variants. While GMTs against Omicron BA.1 weresignificantly lower compared to Wuhan (GMT 160 vs 398), titers againstOmicron BA.2 were also considerably reduced at 211. Thus, triplevaccination induced a similar level of neutralization against the twoOmicron sublineages (FIG. 16 , A) (A. Muik et al., “Neutralization ofSARS-CoV-2 Omicron by BNT162b2 mRNA vaccine-elicited human sera,”Science (New York, N.Y.), 375, 678-680 (2022),doi:10.1126/science.abn7591; C.-W. Tan et al., “Pan-SarbecovirusNeutralizing Antibodies in BNT162b2-Immunized SARS-CoV-1 Survivors,” TheNew England journal of medicine, 385, 1401-1406 (2021),doi:10.1056/NEJMoa2108453; J. Liu et al., “BNT162b2-elicitedneutralization of B.1.617 and other SARS-CoV-2 variants,” Nature, 596,273-275 (2021), doi:10.1038/s41586-021-03693-y; A. Muik et al.,“Neutralization of SARS-CoV-2 lineage B.1.1.7 pseudovirus by BNT162b2vaccine-elicited human sera,” Science (New York, N.Y.), 371, 1152-1153(2021), doi:10.1126/science.abg6105; and Y. Liu et al., “NeutralizingActivity of BNT162b2-Elicited Serum,” The New England journal ofmedicine, 384, 1466-1468 (2021), doi:10.1056/NEJMc2102017).

Omicron BA.1-breakthrough infection had a marked effect on magnitude andbreadth of the neutralizing antibody response of both double- andtriple-vaccinated individuals, with slightly higher pVN₅₀ GMTs observedin the triple-vaccinated individuals (FIG. 16 , A). The pVN₅₀ GMT ofdouble-vaccinated individuals with breakthrough infection againstOmicron BA.1 and BA.2 was more than 100-fold and 35-fold above the GMTsof Omicron BA.1-naïve double-vaccinated individuals. Immune sera fromdouble-vaccinated individuals with breakthrough infection had broadneutralizing activity, with higher pVN₅₀ GMTs against Beta and Deltathan observed in Omicron-naïve triple-vaccinated individuals (GMT 740vs. 222 and 571 vs. 370). The effect of Omicron BA.1-breakthroughinfection on the neutralization of Omicron BA.1 and BA.2 pseudovirus wasless pronounced when looking at triple-vaccinated individuals(approximately 7-fold and 4-fold increased neutralization compared toOmicron-naïve triple-vaccinated individuals). pVN₅₀ GMTs against OmicronBA.1, BA.2 and Delta were 1029, 836 and 1103 in triple-vaccinatedOmicron breakthrough individuals as compared to 160, 211 and 370 in theOmicron-naïve triple-vaccinated. GMTs against all SARS-CoV-2 VOCs,including Beta and Omicron, were close to titers against the Wuhanreference, while noticeably reduced in triple-vaccinated Omicron-naïveindividuals.

Likewise, while sera from vaccinated Omicron-naïve individuals had nodetectable or only poor pVN₅₀ titers against the phylogenetically moredistant SARS-CoV-1, convalescent sera of double- and even more markedlyof triple-vaccinated Omicron infected individuals robustly neutralizedSARS-CoV-1 pseudovirus (FIG. 16 , A and B). Nine out of 18 breakthroughinfected individuals (four double-vaccinated and five triple-vaccinated)had SARS-CoV-1 pVN₅₀ GMTs comparable to or above those against the Wuhanreference in Omicron-naïve double-vaccinated individuals (GMT≥120).

Authentic live SARS-CoV-2 virus neutralization assays conducted withWuhan, Beta, Delta and Omicron BA.1 pseudoviruses also showed similarfindings (FIG. 16 , B). In BNT162b2 double- and triple-vaccinatedindividuals, Omicron BA.1 infection was associated with a stronglyincreased neutralizing activity against Omicron BA.1 with 50% virusneutralization (VN₅₀) GMTs in the same range as against the Wuhan strain(FIG. 16 , B; GMT 493 vs. 381 and GMT 538 vs. 613). Similarly, OmicronBA.1 convalescent double- and triple-vaccinated individuals showedcomparable levels of neutralization against other variants as well(e.g., GMT 493 and 729 against Beta), indicating a wide breadth ofneutralizing activity.

In aggregate, these data demonstrate that SARS-CoV-2 Omicron BA.1breakthrough infection induces neutralization activity of profoundbreadth in vaccine-experienced individuals, a finding further supportedby the calculated ratios of VN₅₀ GMTs against the Wuhan strain andSARS-CoV-2 VOCs (FIG. 16 , C). While double- and to a lesser extent alsotriple-BNT162b2 vaccinated Omicron-naïve individuals displayed markeddifferences in neutralization proficiency against VOCs, neutralizationactivity of Omicron BA.1 convalescent subjects was leveled to almost thesame range of high performance against all variant strains tested.Likewise, Omicron BA.1 breakthrough infection had a similarly broadneutralization augmenting effect in individuals vaccinated with otherapproved COVID-19 vaccines or heterologous regimens (FIG. 19 ; Table20).

TABLE 20 Individuals vaccinated with other approved COVID-19 vaccines ormixed regimens after subsequent Omicron BA.1 breakthrough infectionPositive test Blood draw Omicron Dose 1-2 Dose 2-3 after last afterpositive Vaccination subtype interval interval vaccination test AZ/BNTn/a 62 N/A 142 46 AZ/BNT n/a 68 N/A 135 45 J&J n/a N/A N/A 161 44 MOD³BA.1 42 172 3 35 MOD² n/a 42 N/A 169 44 J&J/BNT n/a 138 N/A 45 40AZ/BNT/MOD BA.1 68 154 9 43 MOD²/BNT n/a 28 252 22 40 MOD²/BNT n/a 42154 13 42 MOD²/BNT n/a 28 256 45 31 Median: 45 43 n/a, not available;N/A, not applicable; AZ, AstraZeneca AZD1222; BNT, BioNTech/PfizerBNT162b2; J&J, Johnson & Johnson Ad26.COV2.S; MOD, Moderna mRNA-1273;BNT⁴, BNT162b2 four-dose series; MOD², mRNA-1273 two-dose series; MOD³,mRNA-1273 three-dose series

B_(MEM) Cells of BNT162b2 Double- and of Triple-Vaccinated IndividualsBroadly Recognize VOCs and are Further Boosted by Omicron BreakthroughInfection

Next, the phenotype and quantity of SARS-CoV-2 S protein specific Bcells were investigated. Flow cytometry-based B cell phenotyping assayswere used for differential detection of variant-specific Sprotein-binding B cells in bulk PBMCs. All S protein- and RBD-specific Bcells in the peripheral blood were found to be of a B_(MEM) phenotype(B_(MEM); CD^(20high)CD^(38int/neg)), as antigen-specific plasmablastsor naïve B cells were not detected (data not shown). The assaystherefore allowed the differentiation for each of the SARS-CoV-2variants between B_(MEM) cells recognizing the full S protein or its RBDthat is a hotspot for amino acid alterations, and variant-specificantigenic epitopes (FIG. 17 , A).

The overall frequency of antigen-specific B_(MEM) cells varied acrossthe different groups. The frequency of B_(MEM) cells in Omicron-naïvedouble-vaccinated individuals was low at an early time point aftervaccination and increased over time: At 5 months as compared to 3 weeksafter the second BNT162b2 dose, S protein-specific B_(MEM) cells almostquadrupled, RBD-specific ones tripled across all VOCs thereby reachingquantities similar to those observed in Omicron-naïve triple-vaccinatedindividuals (FIG. 17 , B and C).

Double or triple BNT162b2-vaccinated individuals with a SARS-CoV-2Omicron BA.1 breakthrough infection exhibited a strongly increasedfrequency of B_(MEM) cells, which was higher than those of Omicron-naïvetriple-vaccinated individuals (FIG. 17 , B and D).

In all groups, including Omicron-naïve and Omicron infected individuals,B_(MEM) cells against Omicron BA.1 S protein were detectable atfrequencies comparable to those against Wuhan and other tested VOCs(FIG. 17 , B and D), whereas the frequency of B_(MEM) cells againstOmicron BA.1 RBD was slightly lower compared to the other variants (FIG.17 , C and E).

The ratios of RBD protein to S protein binding within the differentgroups was then compared and found to be biased towards S proteinrecognition for the Omicron BA.1 VOC, particularly in the Omicron-naïvegroups (FIG. 17 , F). In the Omicron BA.1-experienced groups this ratiois higher, indicating that an Omicron BA.1 breakthrough infectionimproved Omicron BA.1 RBD recognition.

Omicron BA.1 Breakthrough Infection in BNT162b2 Double- andTriple-Vaccinated Individuals Primarily Boosts B_(MEM) Cells AgainstConserved Epitopes Shared Broadly Between S Proteins of Wuhan and OtherVOCs Rather than Strictly Omicron S-Specific Epitopes.

These findings indicate that Omicron BA.1 infection in vaccinatedindividuals boosts not only neutralizing activity and B_(MEM) cellsagainst Omicron BA.1, but broadly augments immunity against variousVOCs. To investigate the specificity of antibody responses at a cellularlevel, multi-parameter analyses of B_(MEM) cells stained withfluorescently labeled variant-specific S or RBD proteins were performed.

A combinatorial gating strategy was applied to distinguish betweenB_(MEM) cell subsets that could identify only single variant-specificepitopes of Wuhan, Alpha, Delta or Omicron BA.1, versus those that couldidentify any given combination thereof (FIG. 18 , A).

In a first analysis, B_(MEM) cell recognition of Wuhan and Omicron BA.1S and RBD proteins was evaluated (FIG. 18 , B, C, and D). The SARS-CoV-2Omicron BA.1 variant has 37 amino acid alterations in the S proteincompared to the Wuhan parental strain, of which 15 alterations are inthe RBD, an immunodominant target of neutralizing antibodies induced byCOVID-19 vaccines or by SARS-CoV-2 infections.

Staining with full length S proteins showed that the largest proportionof B_(MEM) cells from Omicron-naïve double-vaccinated individuals, andeven more predominantly from triple-vaccinated individuals were directedagainst epitopes shared by both Wuhan and Omicron BA.1 SARS-CoV-2variants. Consistent with the observation that vaccination with BNT162b2can elicit immune responses against wild-type epitopes that do notrecognize the corresponding altered epitopes in the Omicron BA.1 Sprotein (FIG. 18 , B and C), in most individuals a smaller but clearlydetectable proportion of B_(MEM) cells was found that recognized onlyWuhan S protein or RBD. Consistent with the lack of exposure, no B_(MEM)cells binding exclusively to Omicron BA.1 S or RBD protein were detectedin these Omicron-naïve individuals.

In Omicron BA.1 convalescent individuals, frequencies of B_(MEM) cellsrecognizing S protein epitopes shared between Wuhan and Omicron BA.1were significantly higher than in the Omicron-naïve ones (FIG. 18 , Band C). In most of these subjects, a small proportion of exclusivelyWuhan S protein-specific B_(MEM) cells was found, as well as a slightlylower frequency of exclusively Omicron BA.1 variant S protein-specificones.

A similar but slightly different pattern was observed by B cell stainingwith labeled RBD proteins (FIG. 18 , B and D). Again, Omicron BA.1breakthrough infection of double-/triple-vaccinated individuals wasfound to primarily boost B_(MEM) cells reactive with conserved epitopes.A moderate boost of Wuhan-specific reactivities was observed; however,only small populations of Omicron-RBD-specific B_(MEM) cells weredetected in the tested individuals (FIG. 18 , D).

Next, the combinatorial gating approach was used to identify the subsetsof S protein or RBD binding B_(MEM) cells that either bind exclusivelyto Wuhan or Omicron BA.1, or to common epitopes conserved broadly acrossall four variants, Wuhan, Alpha, Delta and Omicron BA.1 (FIG. 18 , E).Across all four study groups, the frequency of B_(MEM) cells recognizingS protein epitopes was found to be conserved across all tested variants,accounting for the largest fraction of the pool of S protein-bindingB_(MEM) cells (FIG. 18 , F, all 4+ve). The S protein of the Wuhan straindoes not have an exclusive amino acid change that distinguishes it fromthe spike proteins of the Alpha, Delta, or Omicron BA.1 VOCs.Accordingly, B_(MEM) cells exclusively recognizing the Wuhan S proteinwere hardly detected in any individual (FIG. 18 , F). In severalindividuals with Omicron BA.1 breakthrough infection, a small proportionof B_(MEM) cells was detected that bound exclusively to Omicron BA.1 Sprotein (FIG. 18 , F), whereas almost none of the individuals displayeda strictly Omicron BA.1 RBD-specific response (FIG. 18 , G).

These findings indicate that SARS-CoV-2 Omicron BA.1 breakthroughinfection in vaccinated individuals primarily expands a broad B_(MEM)cell repertoire against conserved S protein and RBD epitopes, ratherthan inducing large numbers of Omicron-specific B_(MEM) cells.

To further dissect this response, the B_(MEM) subsets directed againstthe RBD were characterized. The combinatorial Boolean gating approachwas used to discern B_(MEM) cells with distinct binding patterns in thespectrum of strictly variant-specific and common epitopes shared byseveral variants. Multiple sequence alignments revealed that the OmicronBA.1 RBD diverges from the RBD sequence regions conserved in Wuhan,Alpha and Delta by 13 single amino acid alterations. All Omicron BA.1convalescent individuals were found to have robust frequencies ofB_(MEM) cells that recognized Wuhan, Alpha as well as the Delta VOCRBDs, but not Omicron BA.1 RBD, while B_(MEM) cells exclusively reactivewith Omicron BA.1 RBD were almost absent in most of those individuals(FIG. 18 , H). B_(MEM) cells that exclusively recognized the OmicronBA.1 and Alpha RBDs, or the Omicron BA.1 and Delta RBDs were also notdetected.

Furthermore, in all individuals two additional subsets of RBD-specificB_(MEM) cells were identified. One subset was characterized by bindingto Wuhan, Alpha and Omicron BA.1, but not Delta, RBD. The otherpopulation exhibited binding to Wuhan and Alpha but not Omicron BA.1 orDelta RBD (FIG. 18 , H). Sequence alignment identified L452R as the onlyRBD mutation unique for Delta that is not shared by the other 3 variantRBDs (FIG. 18 , I top). Similarly, the only RBD site conserved in Wuhanand Alpha but altered in Delta and Omicron BA.1 was found to be T478K(FIG. 18 , I bottom). Both L452R and T478K alterations are known to beassociated with the evasion of vaccine induced neutralizing antibodyresponses. Of note, no B_(MEM) cells were detected in all combinatorialsubgroups in which multiple sequence alignment failed to identify uniqueepitopes in the RBD sequence that satisfied the Boolean selectioncriteria (e.g., Wuhan only or Wuhan and Omicron BA.1, but not Alpha,Delta). These findings indicate that the B_(MEM) cell response againstRBD is driven by specificities induced through prior vaccination withBNT162b2 and not substantially redirected against new RBD epitopesmutated in the Omicron variant after infection.

SUMMARY

SARS-CoV-2 Omicron is a partial immune escape variant with anunprecedented number of amino acid alterations in the S protein at sitesof neutralizing antibody binding, distinguishing it from previouslyreported variants. Recent neutralizing antibody mapping and molecularmodeling studies strongly support the functional relevance of thesealterations, and their importance is confirmed by the observation thatdouble-vaccinated individuals have no detectable neutralizing activityagainst SARS-CoV-2 Omicron.

The findings presented herein show that Omicron BA.1 breakthroughinfection of vaccinated individuals boosts not only neutralizingactivity and B_(MEM) cells against Omicron BA.1 but broadly augmentsimmunity against various VOCs, and also provide insights into how broadimmunity is achieved

The data presented herein indicate that initial exposure to the Wuhanstrain S protein may have shaped the formation of B_(MEM) cells andimprinted against the formation of novel B_(MEM) cell responses againstthe more distinctive epitopes of the Omicron BA.1 variant. Similarobservations have been reported from vaccinated individuals whoexperienced breakthrough infections with the delta variant (K. Röltgenet al., “Immune imprinting, breadth of variant recognition, and germinalcenter response in human SARS-CoV-2 infection and vaccination,” Cell(2022), doi:10.1016/j.cell.2022.01.018.). As demonstrated in the presentExample, Omicron BA.1 breakthrough infection primarily expands a broadB_(MEM) cell repertoire against conserved S protein and RBD epitopes,rather than inducing considerable numbers of strictly Omicron-specificB_(MEM) cells.

Thus, Omicron BA.1 breakthrough infection in double-vaccinatedindividuals leads to expansion of the pre-existing B_(MEM) cell pool,similar to a third dose of booster vaccination. However, there are cleardifferences in the immune response pattern induced by a homologousvaccine booster as compared to an Omicron BA.1 breakthrough infection.Despite the focus of the B cell memory response on conserved epitopes,Omicron BA.1 breakthrough infection leads to a more substantial increasein antibody neutralization titers against Omicron BA.1, as well aspronounced cross-neutralization of both the ancestral and the novel SARSCoV-2 variants. These effects are particularly striking indouble-vaccinated individuals.

Without wishing to be bound by theory, three findings may point topotentially complementary and synergistic mechanisms responsible forthese results:

First, an overall increase of S protein-specific B_(MEM) cells. OmicronBA.1-convalescent double-vaccinated individuals have a higher frequencyof B_(MEM) cells and higher neutralizing antibody titers against allVOCs as compared to triple-vaccinated individuals. That breakthroughinfection elicits a stronger neutralizing antibody response than the 3rdvaccine dose in double-vaccinated individuals is not apparent fromprevious studies describing breakthrough infections with other variants(Evans et al., Science Translational Medicine (2022) 14, eabn8057) andmay be explained by poor neutralization of the Omicron BA.1 variant inthe initial phase of infection, potentially causing a greater orprolonged antigen exposure of the immune system to the altered Sprotein.

Second, a stronger bias on RBD-specific B_(MEM) cell responses. OmicronBA.1 breakthrough infection promotes proportionally more pronouncedboosting of RBD-specific B_(MEM) cells than of B_(MEM) cells thatrecognize S protein-specific epitopes outside the RBD. Therefore,Omicron BA.1-infected individuals have a significantly higher ratio ofRBD/S protein-specific B_(MEM) cells compared to vaccinatedOmicron-naïve individuals. The RBD is a key domain of the S protein thatbinds to the SARS-CoV-2 receptor ACE2 and has multiple neutralizingantibody binding sites in regions that are not affected by Omicron BA.1alterations, e.g., position L452. An increased focus of the immuneresponse on this domain could promote B_(MEM) cells producingneutralizing antibodies against RBD epitopes that are not altered inOmicron BA.1.

Third, the induction of broadly neutralizing antibodies. The majority ofsera from Omicron-BA.1 convalescent but not from Omicron-naïvevaccinated individuals was found to robustly neutralize SARS-CoV-1. Thismay indicate that Omicron BA.1 infection in vaccinated individualsstimulates B_(MEM) cells that form neutralizing antibodies against spikeprotein epitopes conserved in the SARS-CoV-1 and SARS-CoV-2 families. Itwas reported that broadly neutralizing antibodies are present inSARS-CoV-1 infected individuals vaccinated with BNT162b2. Suchpan-Serbecovirus immune responses are thought to be triggered byneutralizing antibodies to highly conserved S protein domains. Thegreater antigenic distance of the Omicron BA.1 spike protein from theother SARS-Cov-2 strains may promote targeting of conserved subdominantneutralizing epitopes as recently described to be located in theC-terminal portion of the spike protein.

In aggregate, these results indicate that despite possible imprinting ofthe immune response by previous vaccination, the preformed B-cell memorypool can be refocused and quantitatively remodeled by exposure toheterologous S proteins to allow neutralization of variants that evade apreviously established neutralizing antibody response.

In conclusion, while the data are based on samples from individualsexposed to the Omicron BA.1 S protein as a result of infection, thefindings presented herein support that a vaccine adapted to the OmicronBA.1 S protein can similarly reshape the B-cell memory repertoire andtherefore can be more beneficial than an extended series of boosterswith the existing Wuhan-Hu-1 spike based vaccines.

Materials and Methods

Recruitment of Participants and Sample Collection

Individuals from the SARS-CoV-2 Omicron-naïve BNT162b2 double-vaccinated(BNT162b2²) and triple-vaccinated (BNT162b2³) cohorts provided informedconsent as part of their participation in a clinical trial (the Phase1/2 trial BNT162-01 [NCT04380701], the Phase 2 rollover trial BNT162-14[NCT04949490], or as part of the BNT162-17 [NCT05004181] trial).Participants from the SARS-CoV-2 Omicron BA.1 convalescent double- andtriple vaccinated cohorts (BNT162b2²+Omi and BNT162b2³+Omi cohorts,respectively) and individuals vaccinated with other approved COVID-19vaccines or mixed regimens with subsequent Omicron BA.1 breakthroughinfection were recruited from University Hospital, Goethe UniversityFrankfurt as part of a research program that recruited patients that hadexperienced Omicron BA.1 breakthrough infection following vaccinationfor COVID-19, to provide blood samples and clinical data for research.Infection with the Omicron BA.1 strain was confirmed withvariant-specific PCR or sequencing, and participants were free ofsymptoms at the time of blood collection.

Sampling timepoints are provided in FIG. 15 .

Serum was isolated by centrifugation 2000×g for 10 minutes andcryopreserved until use. Li-Heparin blood samples were isolated bydensity gradient centrifugation using Ficoll-Paque PLUS (Cytiva) andwere subsequently cryopreserved until use.

VSV-SARS-CoV-2 S Variant Pseudovirus Generation

A recombinant replication-deficient vesicular stomatitis virus (VSV)vector that encodes green fluorescent protein (GFP) and luciferaseinstead of the VSV-glycoprotein (VSV-G) was pseudotyped with SARS-CoV-1spike (S) (UniProt Ref: P59594) and with SARS-CoV-2 S derived fromeither the Wuhan reference strain (NCBI Ref: 43740568), the Alphavariant (mutations: Δ69/70, Δ144, N501Y, A570D, D614G, P681H, T716I,S982A, D1118H), the Beta variant (mutations: L18F, D80A, D215G,Δ242-244, R246I, K417N, E484K, N501Y, D614G, A701V), the Delta variant(mutations: T19R, G142D, E156G, Δ157/158, K417N, L452R, T478K, D614G,P681R, D950N) the Omicron BA.1 variant (mutations: A67V, Δ69/70, T95I,G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F,K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y,Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H,N969K, L981F) or the Omicron BA.2 variant (mutations: T19I, Δ24-26,A27S, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S,K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G,H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K) according to publishedpseudotyping protocols (M. Berger Rentsch, G. Zimmer, A vesicularstomatitis virus replicon-based bioassay for the rapid and sensitivedetermination of multi-species type I interferon. PloS one. 6, e25858(2011), doi:10.1371/journal.pone.0025858).

In brief, HEK293T/17 monolayers (ATCC® CRL-11268™) cultured inDulbecco's modified Eagle's medium (DMEM) with GlutaMAX™ (Gibco)supplemented with 10% heat-inactivated fetal bovine serum (FBS[Sigma-Aldrich]) (referred to as medium) were transfected with Sangersequencing-verified SARS-CoV-1 or variant-specific SARS-CoV-2 Sexpression plasmid with Lipofectamine LTX (Life Technologies) followingthe manufacturer's instructions. At 24 hours VSV-G complemented VSVΔGvector. After incubation for 2 hours at 37° C. with 7.5% CO₂, cells werewashed twice with phosphate buffered saline (PBS) before mediumsupplemented with anti-VSV-G antibody (clone 8G5F11, Kerafast Inc.) wasadded to neutralize residual VSV-G-complemented input virus.VSV-SARS-CoV-2-S pseudotype-containing medium was harvested hours afterinoculation, passed through a 0.2 μm filter (Nalgene) and stored at −80°C. The pseudovirus batches were titrated on Vero 76 cells (ATCC®CRL-1587™) cultured in medium. The relative luciferase units induced bya defined volume of a Wuhan spike pseudovirus reference batch previouslydescribed in Muik et al. (Muik et al., “Neutralization of SARS-CoV-2lineage B.1.1.7 pseudovirus by BNT162b2 vaccine-elicited human sera.Science (New York, N.Y.). 371, 1152-1153 (2021),doi:10.1126/science.abg6105”) that corresponds to an infectious titer of200 transducing units (TU) per mL, was used as a comparator. Inputvolumes for the SARS-CoV-2 variant pseudovirus batches were calculatedto normalize the infectious titer based on the relative luciferase unitsrelative to the reference.

Pseudovirus Neutralization Assay

Vero 76 cells were seeded in 96-well white, flat-bottom plates (ThermoScientific) at 40,000 cells/well in medium 4 hours prior to the assayand cultured at 37° C. with 7.5% CO₂. Each serum was serially diluted2-fold in medium with the first dilution being 1:5 (Omicron naïvedouble- and triple BNT162b2 vaccinated; dilution range of 1:5 to1:5,120) or 1:30 (double- and triple BNT162b2 vaccinated aftersubsequent Omicron breakthrough infection; dilution range of 1:30 to1:30,720). VSV-SARS-CoV-2-S/VSV-SARS-CoV-1-S particles were diluted inmedium to obtain 200 TU in the assay. Serum dilutions were mixed 1:1with pseudovirus (n=2 technical replicates per serum per pseudovirus)for 30 minutes at room temperature before being added to Vero 76 cellmonolayers and incubated at 37° C. with 7.5% CO₂ for 24 hours.Supernatants were removed and the cells were lysed with luciferasereagent (Promega). Luminescence was recorded on a CLARIOstar® Plusmicroplate reader (BMG Labtech), and neutralization titers werecalculated as the reciprocal of the highest serum dilution that stillresulted in 50% reduction in luminescence. Results were expressed asgeometric mean titers (GMT) of duplicates. If no neutralization wasobserved, an arbitrary titer value of half of the limit of detection[LOD] was reported.

Live SARS-CoV-2 Neutralization Assay

SARS-CoV-2 virus neutralization titers were determined by amicroneutralization assay based on cytopathic effect (CPE) at VisMederiS.r.l., Siena, Italy. In brief, heat-inactivated serum samples fromparticipants were serially diluted 1:2 (starting at 1:10) and incubatedfor 1 hour at 37° C. with 100 TCID50 of live Wuhan-like SARS-CoV-2 virusstrain 2019-nCOV/ITALY-INMI1 (GenBank: MT066156), Beta virus strainHuman nCoV19 isolate/England ex-SA/HCM002/2021 (mutations: D80A, D215G,Δ242-244, K417N, E484K, N501Y, D614G, A701V), sequence-verified Deltastrain isolated from a nasopharyngeal swab (mutations: T19R, G142D,E156G, Δ157/158, L452R, T478K, D614G, P681R, R682Q, D950N) or OmicronBA.1 strain hCoV-19/Belgium/rega-20174/2021 (mutations: A67V, Δ69/70,T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D, S371L, S373P,S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R,N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K,Q954H, N969K, L981F) to allow any antigen-specific antibodies to bind tothe virus. The 2019-nCOV/ITALY-INMI1 strain S protein is identical insequence to the wild-type SARS-CoV-2 S (Wuhan-Hu-1 isolate). Vero E6(ATCC® CRL-1586™) cell monolayers were inoculated with the serum/virusmix in 96-well plates and incubated for 3 days (2019-nCOV/ITALY-INMI1strain) or 4 days (Beta, Delta and Omicron BA.1 variant strain) to allowinfection by non-neutralized virus. The plates were observed under aninverted light microscope and the wells were scored as positive forSARS-CoV-2 infection (i.e., showing CPE) or negative for SARS-CoV-2infection (i.e., cells were alive without CPE). The neutralization titerwas determined as the reciprocal of the highest serum dilution thatprotected more than 50% of cells from CPE and reported as GMT ofduplicates. If no neutralization was observed, an arbitrary titer valueof 5 (half of the limit of detection [LOD]) was reported.

Detection and Characterization of SARS-CoV-2-Specific B Cells with FlowCytometry

Spike/RBD-specific B cells were detected using recombinant, biotinylatedSARS-CoV-2 Spike (Acro Biosystems: Wuhan—SPN-C82E9, Alpha—SPN-C82E5,Delta—SPN-C82Ec, Omicron—SPN-C82Ee) and RBD (Acro Biosystems:Wuhan—SPD-B28E9, Alpha—SPD-C82E6, Delta—SPD-C82Ed, Omicron—SPD-C82E4)proteins. Recombinant Spike and RBD proteins were tetramerized withfluorescently labeled Streptavidin (BioLegend, BD Biosciences) in a 4:1molar ratio for 1 h at 4° C. in the dark. Afterwards samples were spundown for 10 min at 4° C. to remove eventual precipitates.

For flow cytometric analysis, PBMCs were thawed and 5×10⁶ cells persample were seeded into 96 U-bottom plates. Cells were blocked forFc-receptor-binding (Human BD Fc Block™, BD Biosciences) and staturedwith free biotin (D-Biotin, Invitrogen, 1 μM) in flow buffer (DPBS(Gibco) supplemented with 2% FBS (Sigma), 2 mM EDTA (Sigma-Aldrich)) for20 min at 4° C. Cells were washed and labeled with BCR bait tetramerssupplemented with free Biotin in flow buffer (D-Biotin, Invitrogen, 2μg/ml) for 1 h at 4° C. in the dark (2 μg/ml for Spike and 0.25 μg/mlfor RBD proteins). Cells were washed with flow buffer and stained forviability (Fixable Viability Dye eFluor™ 780, eBioscience) and surfacemarkers (CD3—clone: UCHT1 (BD Biosciences), CD4—clone: SK3 (BDBiosciences), CD185 (CXCR5)—clone: RF8B2 (BioLegend), CD279(PD-1)—clone: EH12.1 (BD Biosciences), CD278 (ICOS)—clone: C398.4A(BioLegend), CD19—clone: SJ25C1 (BD Biosciences), CD20—clone: 2H7 (BDBiosciences), CD21—clone: B-ly4 (BD Biosciences), CD27—clone: L128 (BDBiosciences), CD38—clone: HIT2 (BD Biosciences), CD11c—clone: S-HCL-3(BD Biosciences), CD138—clone: M115 (BD Biosciences), IgG—clone: G18-145(BD Biosciences), IgM—clone: G20-127 (BD Biosciences), IgD—clone: IA6-2(BD Biosciences), CD14—clone: MϕP9 (BD Biosciences, dump channel),CD16—clone: 3G8 (BD Biosciences, dump channel)) in flow buffersupplemented with Brilliant Stain Buffer Plus (BD Biosciences, accordingto the manufacturer's instructions) for 20 min at 4° C. Samples werewashed and fixed with BDTM Stabilizing Fixative (BD Biosciences,according to the manufacturer's instructions) prior to data acquisitionon a BD Symphony A3 flow cytometer. FCS 3.0 files were exported from BDDiva Software and analyzed using FlowJo software (Version 10.7.1.).

Debris and doublets were discriminated via FSC/SSC. Then dead cells andmonocytes (CD14, CD16—Viability/Dump channel) were excluded. CD19positive B cells were analyzed for IgD and CD27 expression, therebynaïve B cells were discriminated as IgD⁺ cells with the Boolean ‘makenon-gate’ function. Within non-naïve B cells Plasmablasts (CD38^(high),CD20^(low)) and memory B cells (B_(MEM)S CD38^(int/low)CD20^(high)) weredistinguished. B_(MEM) cells were analyzed for B cell bait binding.SARS-CoV-2 Spike reactivities were assessed by gating on each Spike/RBDvariant tested by plotting against the CD20 signal. Bait gates wereoverlayed onto total B_(MEM) cells and displayed as N×N-Plots for thefour bait channels.

Statistical Analysis

The statistical method of aggregation used for the analysis of antibodytiters is the geometric mean and for the ratio of SARS-CoV-2 VOC titerand Wuhan titer the geometric mean and the corresponding 95% confidenceinterval. The use of the geometric mean accounts for the non-normaldistribution of antibody titers, which span several orders of magnitude.The Friedman test with Dunn's correction for multiple comparisons wasused to conduct pairwise signed-rank tests of group geometric meanneutralizing antibody titers with a common control group. Flowcytometric frequencies were analyzed with and tables were exported fromFlowJo software (Version 10.7.1.). Statistical analysis of cumulativememory B cell frequencies was the mean and standard errors of the mean(SEM). All statistical analyses were performed using GraphPad Prismsoftware version 9.

Example 8: Induced Antibody Response of Vaccines Encoding a SARS-CoV-2 SProtein from an Omicron Variant

To test the efficacy of an RNA vaccine encoding a SARS-CoV-2 S proteincomprising one or more mutations characteristic of an Omicron variant,subjects previously administered a primary regimen comprising two dosesof 30 ug of RNA encoding a SARS-CoV-2 S protein from a Wuhan strain(e.g., BNT162b2), and a booster regimen comprising a dose of 30 ug ofRNA encoding a SARS-CoV-2 S protein from a Wuhan strain (i.e., a Wuhanspecific booster, e.g., BNT162b2) were administered a further boosterdose comprising either (i) 30 ug of RNA encoding an SARS-CoV-2 S proteinfrom a Wuhan strain (e.g., BNT162b2), or (ii) 30 ug of RNA encoding aSARS-CoV-2 S protein comprising mutations that are characteristic of anOmicron BA.1 variant (i.e., an Omicron BA.1-specific booster, e.g., RNAencoding a SARS-CoV-2 S protein comprising an amino acid sequence of SEQID NO: 49, and/or comprising a nucleotide sequence of SEQ ID NOs: 50and/or 51) (the dose administered as part of the second booster regimenis referred to as a “4th dose” in the figures). Sera was collected fromsubjects at the time of administering the second booster regimen and onemonth afterwards.

Neutralization antibody titers were determined using a Fluorescent FocusReduction Neutralization Test (“FFRNT”). Suitable FFRNT assays are knownin the art, and include, e.g., the assays described in Zou J, Xia H, XieX, et al. “Neutralization against Omicron SARS-CoV-2 from previousnon-Omicron infection,” Nat Commun 2022; 13:852, the contents of whichis incorporated by reference herein in its entirety. Additionalexemplary neutralization assays include those described in the previousexamples, as well as those described in Bewley, Kevin R., et al.“Quantification of SARS-CoV-2 neutralizing antibody by wild-type plaquereduction neutralization, microneutralization and pseudotyped virusneutralization assays.” Nature Protocols 16.6 (2021): 3114-3140. Asshown in FIG. 20 , A, subjects administered a second booster regimencomprising a dose of RNA encoding a SARS-CoV-2 S protein comprisingmutations characteristic of an Omicron BA.1 variant exhibitedsignificant increases in concentrations of neutralization antibodiesagainst an Omicron BA.1 variant, as compared to subjects administered asecond booster regimen comprising a dose of RNA encoding a SARS-CoV-2 Sprotein of a Wuhan strain. Specifically, subjects administered anOmicron BA.1 specific booster exhibited a GMR that was 1.79-fold higherand a GMFR that was 2.31 fold higher than that observed in subjectsadministered a fourth dose of RNA encoding a SARS-CoV-2 S protein from aWuhan strain. The superior immune response induced by an OmicronBA.1-specific booster against an Omicron BA.1 variant was furtherincreased in subjects previously infected with SARS-CoV-2 (as determinedby an antigen test) or currently infected with SARS-CoV-2 (as determinedby PCR). See FIG. 20 , B, which shows that a subject populationincluding previously and/or currently infected subjects exhibited a GMRthat is 2.94 fold higher, and a GMFR ratio that is 1.97 fold higher thatobserved in a subject population comprising previously and/or currentlyinfected subjects administered an RNA vaccine encoding a SARS-CoV-2 Sprotein from a Wuhan strain.

Pseudovirus neutralization assays were also performed using apseudovirus comprising a SARS-CoV-2 S protein of a Wuhan strain, usingthe same sera samples discussed above. Subjects administered RNAencoding a SARS-CoV-2 S protein from a Wuhan strain (e.g., BNT162b2)exhibited titers of neutralization antibodies that were similar to thoseobserved in subjects administered an Omicron BA.1-specific booster,demonstrating that the two vaccines are at least similarly effective intheir ability to induce an antibody response against a Wuhan strain. SeeFIG. 20C, which shows that the GMR and GMFR observed in subjectsadministered a Wuhan specific booster (e.g., BNT162b2) is similar tothat observed in subjects administered an Omicron BA.1 specific booster(OMI). In subjects previously infected with SARS-CoV-2 (e.g., asdetermined by an antigen assay) or currently infected with SARS-CoV-2(e.g., as determined by a PCR assay), subjects administered an OmicronBA.1 specific booster demonstrated an improved immune response ascompared to subjects administered a booster specific for a Wuhan strain.See FIG. 20D, which shows that the GMR for subjects administered anOmicron BA.1 specific booster is about 1.4 fold that of subjectsadministered a Wuhan specific booster. Subjects administered an OmicronBA.1 specific booster also demonstrated a superior immune responseagainst a delta variant in pseudovirus neutralization assays. See FIG.20E, showing that the GMFR for subjects administered an OmicronBA.1-specific booster is about 1.20 fold higher than that observed insubjects administered a Wuhan specific booster. The superior immuneresponse induced by an Omicron BA.1-specific booster against a deltavariant was further increased in sera from subjects previously and/orcurrently infected with SARS-CoV-2. See FIG. 20F.

Example 9: Immunogenicity Study of Vaccines Encoding S Proteins ofSARS-CoV-2 Variants in Vaccine-Naïve Subjects

To test the immunogenicity of various variant specific vaccines invaccine naïve subjects, vaccine naïve mice were immunized twice with (a)saline (negative control), (b) an RNA vaccine encoding a SARS-CoV-2 Sprotein from a Wuhan strain, (c) an RNA vaccine encoding a SARS-CoV-2 Sprotein having mutations characteristic of an Omicron BA.1 variant(Omi), (d) an RNA vaccine encoding a SARS-CoV-2 S protein havingmutations characteristic of a delta variant (Delta), (e) a bivalentvaccine comprising RNA encoding a SARS-CoV-2 S protein from a Wuhanstrain and a SARS-CoV-2 S protein comprising mutations characteristic ofan Omicron BA.1 variant (b2+Omi), and (f) a bivalent vaccine comprisingRNA encoding a SARS-CoV-2 S protein having mutations characteristic of adelta variant and RNA encoding a SARS-CoV-2 S protein having mutationscharacteristics of an Omicron BA.1 variant (Delta+Omi). Theimmunogenicity of the RNA vaccines was investigated by focusing on theantibody immune response.

Sera was obtained 7 days after immunization, and analyzed using apseudovirus neutralization assay (e.g., the assay described in Example2), using pseudoviruses comprising a SARS-CoV-2 S protein from a Wuhanstrain, a SARS-CoV-2 S protein comprising mutations characteristic of abeta variant, a SARS-CoV-2 S protein comprising mutations characteristicof a delta variant, or a SARS-CoV-2 S protein comprising mutationscharacteristic of an Omicron BA.1 variant. As shown in FIG. 21 ,bivalent vaccines were found to elicit the broadest immune response invaccine naïve mice.

Example 10: Induced Antibody Response of Vaccines Encoding a SARS-CoV-2S Protein Comprising One or More Mutations Characteristic of a BetaVariant in Subjects Previously Administered an RNA Vaccine Encoding aSARS-CoV-2 S Protein from a Wuhan Strain

To test the efficacy of an RNA vaccine encoding a SARS-CoV-2 S proteincomprising one or more mutations characteristic of a Beta variant,subjects previously administered a primary regimen comprising two doseseach of 30 ug of RNA encoding a SARS-CoV-2 S protein from a Wuhan strain(in the present example, BNT162b2 (SEQ ID NO: 20)), were administeredtwo booster doses, each comprising 30 ug of RNA encoding a SARS-CoV-2 Sprotein comprising one or more mutations characteristic of a Betavariant (referred to hereafter as a Beta-specific vaccine). In thepresent Example, construct RBP020.11 was administered as theBeta-specific vaccine. While in the present Example, the two boosterdoses were administered approximately one month apart, in someembodiments, the two booster doses can be administered at least 3 weeksapart, at least 4 weeks apart, at least 5 weeks apart, at least 6 weeksapart, at least 7 weeks apart, at least 8 weeks apart, or longer (e.g.,in accordance with exemplary dosing regimens as described herein).

Sera were collected from subjects before administration of BNT162b2, onemonth after administering two primary doses of BNT162b2, one month afteradministering a first dose of a Beta-specific vaccine, and one monthafter administering a second dose of a Beta-specific vaccine.Neutralization antibody titers against a pseudovirus comprising aSARS-CoV-2 S protein of a Wuhan strain or a SARS-CoV-2 S proteincomprising one or more mutations characteristic of a Beta variant weremeasured using a pseudovirus neutralization assay (results shown in FIG.22 ). Subjects exhibited an increase in neutralization antibody titersagainst both a Wuhan strain of SARS-CoV-2 and a Beta variant followingadministration of the third and fourth doses of a Beta-specific vaccine.

Example 11: Induced Antibody Response of Vaccines Encoding a SARS-CoV-2S Protein Comprising One or More Mutations Characteristic of a BetaVariant in Vaccine Naïve Subjects

To test the efficacy of an RNA vaccine encoding a SARS-CoV-2 S proteincomprising one or more mutations characteristic of a Beta variant invaccine naïve subjects, subjects who had not previously beenadministered a SARS-CoV-2 vaccine, and did not show evidence of prior orcurrent infection with SARS-CoV-2 (e.g., as assessed by an antibody testand/or a PCR test) were administered two doses each of 30 ug of RNAencoding a SARS-CoV-2 S protein comprising one or more mutationscharacteristic of a Beta variant (in the present example, RBP020.11).Sera was collected one month after administration of a second dose, andneutralization antibody titers were measured using a viralneutralization assay, using viral particles comprising either aSARS-CoV-2 S protein from a Wuhan strain or a SARS-CoV-2 S proteinhaving one or more mutations characteristic of a Beta variant. Tables 15and 16, below, show the results for the neutralization assay againstBeta variant (results for the neutralization assay against a Wuhanstrain are not shown). As shown in the tables, compared to vaccine-naïvesubjects administered two doses of an RNA vaccine encoding a SARS-CoV-2S protein from a Wuhan strain (in the present Example, BNT162b2), an RNAvaccine encoding a SARS-CoV-2 S protein having mutations characteristicof a Beta variant was found to induce a significantly stronger antibodyresponse against a Beta variant.

TABLE 21 Geometric Mean Fold Rise (GMFR) of titers of neutralizationantibodies, from before Dose 1 to each subsequent time points, inBNT162b2-naïve subjects without evidence of infection up to 1 monthafter dose 2 and administered BNT162b2 or an RNA vaccine encoding aSARS-CoV-2 S protein having one or more mutations characteristic of aBeta variant Vaccine Group (as Assigned/Randomized)^(a) Dose/BNT162b2_(SA) BNT162b2 Sampling (30 μg) (30 μg) Time GMFR^(d) GMFR^(d)Assay Point^(b) n^(c) (95% CI^(d)) n^(c) (95% CI^(d)) SARS-CoV-2 2/1Month 272 40.0 304 8.3 neutralization (36.2, 44.2) (7.4, 9.2) assay - SAvariant - NT50 Abbreviations: GMFR = geometric mean fold rise; LLOQ =lower limit of quantitation; N-binding = SARS-CoV-2nucleoprotein-binding; NAAT = nucleic acid amplification test; NE = notestimable; NT50 = 50% neutralizing titer; SA = South Africa (Betavariant); SARS-CoV-2 = severe acute respiratory syndrome coronavirus 2.Note: Subjects who had no serological or virological evidence (up to 1month after receipt of Dose 2) of past SARS-CoV-2 infection (i.e.,N-binding antibody [serum] negative at Dose 1 visit, 1-month post-Dose 2visit, SARS-CoV-2 not detected by NAAT[nasal swab] at Dose 1 visit, Dose2 visit, and negative NAAT[nasal swab] at any unscheduled visit up to 1month after Dose 2) were included in the analysis. ^(a)Subjects in theBNT162b2_(SA)/Beta-specific vaccine (30 μg) vaccine group were notrandomized. ^(b)Protocol-specified timing for blood sample collection.^(c)n = Number of subjects with valid and determinate assay results forthe specified assay at both prevaccinationtime point and the givendose/sampling time point. ^(d)GMFRs and 2-sided 95% CIs were calculatedby exponentiating the mean logarithm of fold rises and the correspondingCIs (based on the Student t distribution). Assay results below theLLOQwere set to 0.5 × LLOQ in the analysis.

TABLE 22 Geometric Mean Titers (GMT) of neutralization antibodiesmeasured in vaccine- naïve subjects without evidence of infection up to1 month after dose 2 and administered BNT162b2 or an RNA vaccineencoding a SARS-CoV-2 S protein having one or more mutationscharacteristic of a Beta variant Vaccine Group (asAssigned/Randomized)^(a) BNT162b2_(SA) (30 μg) BNT162b2 (30 μg)Dose/Sampling GMT^(d) GMT^(d) Assay Time Point^(b) n^(c) (95% CI^(d))n^(c) (95% CI^(d)) SARS-CoV-2 1/Prevax   272 34.0 304 33.0neutralization assay - (32.6, 35.3) (33.0, 33.0) SA variant - NT50(titer) 2/1 Month 272 1358.0 304 273.1 (1242.2, 1484.5) (245.6, 303.7)Abbreviations: GMT = geometric mean titer; LLOQ = lower limit ofquantitation; N-binding = SARS-CoV-2 nucleoprotein-binding; NAAT =nucleic acid amplification test; NE = not estimable; NT50 = 50%neutralizing titer; Prevax = prevaccination; SA = South Africa(Beta-variant); SARS-CoV-2 = severe acute respiratory syndromecoronavirus 2. Note: Subjects who had no serological or virologicalevidence (up to 1 month after receipt of Dose 2) of past SARS-CoV-2infection (ie, N-binding antibody [serum] negative at Dose 1 visit,1-month post-Dose 2 visit, SARS-CoV-2 not detected by NAAT[nasal swab]at Dose 1 visit, Dose 2 visit, and negative NAAT[nasal swab] at anyunscheduled visit up to 1 month after Dose 2) were included in theanalysis. ^(a)Subjects in the BNT162b2_(SA)/Beta-specific vaccine (30μg) vaccine group were not randomized. ^(b)Protocol-specified timing forblood sample collection. ^(c)n = Number of subjects with valid anddeterminate assay results for the specified assay at the givendose/sampling time point. ^(d)GMTs and 2-sided 95% CIs were calculatedby exponentiating the mean logarithm of the titers and the correspondingCIs (based on the Student t distribution). Assay results below theLLOQwere set to 0.5 × LLOQ.

Example 12: Induced Antibody Response and Reactogenecity of BNT162b2 orOmicron BA.1-Specific Vaccine as Monovalent, Bivalent and High Dose inParticipants 55+ Years of Age

To test the efficacy and safety of (i) higher doses of RNA vaccines(e.g., as described herein), (ii) RNA vaccines encoding a SARS-CoV-2 Sprotein having one or more mutations characteristic of an Omicron BA.1variant (an Omicron BA.1-specific vaccine), and (iii) a bivalent vaccinecomprising an RNA encoding a SARS-CoV-2 S protein from a Wuhan variantand RNA encoding a SARS-CoV-2 S protein having one or more mutationscharacteristic of an Omicron BA.1 variant, subjects previouslyadministered at least one dose of an RNA vaccine encoding a SARS-CoV-2 Sprotein of a Wuhan strain were administered one of several booster doses(e.g., as described herein). Specifically, subjects who had previouslybeen administered two doses of ug of an RNA vaccine encoding aSARS-CoV-2 S protein from a Wuhan strain (in the present example,BNT162b2), and a third dose of 30 ug of RNA encoding a SARS-CoV-2 Sprotein of a Wuhan strain (also BNT162b2 in the present example), wereadministered a fourth dose comprising:

-   -   (a) 30 ug of RNA encoding a SARS-CoV-2 S protein from a Wuhan        strain,    -   (b) 60 ug of RNA encoding a SARS-CoV-2 S protein from a Wuhan        strain,    -   (c) 30 ug of an Omicron BA.1-specific vaccine,    -   (d) 60 ug of an Omicron BA.1-specific vaccine,    -   e) 30 ug of a bivalent RNA vaccine (Omicron BA.1-adapted        bivalent vaccine), comprising 15 ug of RNA encoding a SARS-CoV-2        S protein from a Wuhan strain and 15 ug of RNA encoding a        SARS-CoV-2 S protein comprising mutations characteristic of an        Omicron BA.1 variant, or    -   (f) 60 ug of a bivalent RNA vaccine (Omicron BA.1-adapted        bivalent vaccine), comprising 30 ug of RNA encoding a SARS-CoV-2        S protein from a Wuhan strain and 30 ug of RNA encoding a        SARS-CoV-2 S protein comprising mutations characteristic of an        Omicron BA.1 variant.

In the present example, for the fourth dose, the RNA encoding aSARS-CoV-2 S protein from a Wuhan variant was BNT162b2, and the RNAencoding a SARS-CoV-2 S protein having mutations characteristic of anOmicron BA.1 variant comprised the nucleotide sequence of SEQ ID NO: 51.

Sera samples were collected at the time of administering the 4th doseand 7 days afterward, and tested for neutralization antibody titersagainst a viral particle comprising a SARS-CoV-2 S protein from a Wuhanstrain, or a SARS-CoV-2 S protein comprising mutations characteristic ofa Delta variant or an Omicron BA.1 variant.

Neutralization antibody titers were determined using a Fluorescent FocusReduction Neutralization Test (“FFRNT”). Suitable FFRNT assays are knownin the art, as discussed in Example 8. The neutralization responses areshown in FIG. 23 .

As shown in FIG. 23 (A) subjects administered a fourth dose of 30 ug ofan Omicron BA.1-specific vaccine exhibited an increase in neutralizationantibodies against an Omicron BA.1 variant as compared to subjectsadministered a fourth dose of 30 ug of BNT162b2. Administering 60 ug ofRNA increased neutralization responses both for BNT162b2 and an OmicronBA.1-specific vaccine, with 60 ug of an Omicron BA.1-specific vaccineshowing a stronger immune response against an Omicron BA.1 variant. Asshown in FIG. 23 (B), similar effects were observed in a population thatincluded subjects previously or currently infected with SARS-CoV-2(e.g., as determined by an antibody test and a PCR test, respectively).

FIG. 23 (C-D) provides data for neutralization responses against a Wuhanstrain of SARS-CoV-2 in a population of subjects excluding subjectspreviously or currently infected with SARS-CoV-2 (FIG. 23(C)) and apopulation of subjects including these subjects (FIG. 23(D)).

FIG. 23 (E-F) provides data for neutralization responses against a Deltavariant in a population of subjects excluding subjects previously orcurrently infected with SARS-CoV-2 (FIG. 23(E)) and a population ofsubjects including these subjects (FIG. 23(F)).

FIG. 23 (G) shows neutralization responses as compared to subjectsadministered a 4th dose of 30 ug of BNT162b2. As can be seen in thetable, an Omicron BA.1-specific vaccine induced a strong responseagainst an Omicron BA.1 variant, and responses that were at leastcomparable to that of BNT162b2 for other variants. A bivalent vaccine(Omicron BA.1-adapted bivalent vaccine) produced a strong immuneresponse against each SARS-CoV-2 variant tested, both at 30 ug and 60 ugdoses.

Reactogenicity of the tested 4^(th) doses was also monitored in patientsfor 7 days following administration of the 4^(th) dose. FIG. 24 (A)shows local immune responses observed in subjects of different groups asindicated. As can be seen in the figure, 60 ug doses of an Omicron BA.1specific vaccine and a bivalent vaccine were found to be more likely toproduce pain at the injection site, as compared to that observed withother tested booster doses; however, the pain was rated as mild ormoderate for both doses. Redness and swelling responses were low andcomparable at each dose tested.

FIG. 24 (B) shows systemic immune responses observed in subjects ofdifferent groups as indicated. Systemic responses (as characterized byfever, fatigue, headache, chills, vomiting, diarrhea, muscle pain, orjoint pain) were similar for each dose, while fatigue trended higherwith the 60 ug doses.

The immune responses and reactogenicity of Omicron BA.1-adapted vaccines(monovalent and bivalent vaccines as described in this Example) as abooster dose are also confirmed in a Phase 2/3 trial in over 1,000participants 56 years of age and older. The geometric mean titers (GMTs)of each of the tested doses, as well as the geometric mean ratios (GMRs)as compared to subjects administered a 4th dose of 30 ug of BNT162b2 areshown in Table 23, below

TABLE 23 Neutralization Responses in Subjects Administered 30 ug or 60ug of a monovalent or bivalent Omicron BA.1-specific vaccine. Vaccinegroup/BNT162b2 30 ug Vaccine GMT (95% CI) GMR Met Superiority Group n 1M post-dose (95% CI) (Y/N) BNT162b2 163 455.8 30 ug (365.9, 567.6)BNT162b2 169 1014.5 2.23 Y OMI 30 ug (825.6, 1246.7) (1.65, 3.00)BNT162b2 174 1435.2 3.15 Y OMI 60 ug (1208.1, 1704.8) (2.38, 4.16)Bivalent 178 711.0 1.56 Y OMI 30 ug (588.3, 829.2) (1.17, 2.08) Bivalent175 900.1 1.97 Y OMI 60 ug (726.3, 1115.6) (1.45, 2.68)

The Omicron BA.1-adapted vaccines (monovalent or bivalent; and 30 ug or60 ug) given as a booster dose elicited substantially higherneutralizing antibody responses against Omicron BA.1 when compared tothat induced by BNT162b2 (encoding a SARS-CoV-2 S protein from a Wuhanstrain). The pre-specified criterion for simple superiority was measuredby the ratio of neutralizing geometric mean titers (GMR) with the lowerbound of the 95% confidence interval >1. The pre-specified criterion forsuper superiority was measured by the ratio of neutralizing geometricmean titers (GMR) with the lower bound of the 95% confidenceinterval >1.5. Based on these criteria, each of the tested doses wasshown to be superior to ug of BNT162b2, and administering 30 ug or 60 ugof BNT162b2 OMI was shown to exhibit super superiority as compared to 30ug of BNT162b2.

Seroresponse rates are summarized in Table 24, below. As shown in thebelow table, each of the tested doses was found to be non-inferior toBNT162b2 30 ug.

TABLE 24 Omicron BA.1 seroresponse rates Seroresponse difference in %(relative to BNT162b2 30 ug group) (95% CI) Met Non- Vaccine n 1 M %Inferiority Group N (%) post-dose (95% CI) (Y/N)¹ BNT162b2 149  85(57.0) (48.7, 65.1) — — 30 ug BNT162b2 163 125 (76.7) (69.4, 82.9) 19.6Y OMI 30 ug (9.3, 29.7) BNT162b2 166 143 (86.1) (79.9, 91.0) 29.1 Y OMI60 ug (19.4, 38.5) Bivalent 169 121 (71.6) (64.2, 78.3) 14.6 Y OMI 30 ug(4.0, 24.9) Bivalent 162 110 (67.9) (60.1, 75.0) 10.9 Y OMI 60 ug (0.1,21.4) ¹Non-inferiority criterion: the lower bound of the 95% confidenceinterval is >−5%

Following the collection of the data summarized in Table 24, datacontinued to be collected on subjects administered an Omicron monovalentvaccine. The further data is summarized in Table 25, below, and confirmsthe findings shown in Table 24.

TABLE 25 Further data on Omicron BA.1 seroresponse rates Vaccine group(as randomized) BNT162b2 OMI (30 ug) BNT162b2 (30 ug) DifferenceDose/sampling n^(b) (%) n^(b) (%) %^(d) Assay time point^(a) N^(a) (95%CI^(c)) N^(a) (95% CI^(c)) (95% CI^(e)) SARS-CoV-2 1/1 month 206 127(61.7) 226 91 (40.3) 21.4 neutralization assay - (54.6, 68.3 (33.8,47.0) (12.0, 30.4) Omicron BA.1 - NT₅₀ (titre) Abbreviations: NT₅₀ = 50%neutralizing titer; SARS-CoV-2 = severe acute respiratory syndromecoronavirus 2. Note: Seroresponse is defined as achieving a ≥4-fold risefrom baseline (before the first dose of study vaccination). If thebaseline measurement is below the LLOQ, the postvaccination measure of≥4 × LLOQ is considered seroresponse. Note: Participants who had noserological or virological evidence (prior to the 1-month post-firststudy vaccination blood sample collection) of past SARS-CoV-2 infection(i.e., N-binding antibody [serum] negative at the first studyvaccination and the 1-month post-first study vaccination visits,negative NAAT [nasal swab] at the first study vaccination visit, and anyunscheduled visit prior to the 1-month post-first study vaccinationblood sample collection) and had no medical history of COVID-19 wereincluded in the analysis.

-   -   a. N=number of participants with valid and determinate assay        results for the specified assay at both the prevaccination time        point and the given sampling time point. This value is the        denominator for the percentage calculations.    -   b. n=Number of participants with seroresponse for the given        assay at the given sampling time point.    -   c. Exact 2-sided CI based on the Clopper and Pearson method.    -   d. Difference in proportions, expressed as a percentage        (monovalent Omicron BA.1 [30 mcg]-Comirnaty [30 mcg]).    -   e. 2-sided CI based on the Miettinen and Nurminen method for the        difference in proportions, expressed as a percentage.

Changes in titers for each dose before administration of a 4th boosterdose and 1 month after administration of a 4th booster dose are shown inFIG. 25 . As shown in FIG. 25 , one month after administration, abooster dose of the Omicron BA.1-adapted monovalent vaccine (30 μg and60 μg) increased neutralizing geometric mean titers (GMT) againstOmicron BA.113.5 and 19.6-fold above pre-booster dose levels, while abooster dose of the Omicron BA.1-adapted bivalent vaccine (30 μg and 60μg) conferred a 9.1 and 10.9-fold increase in neutralizing GMTs againstOmicron BA.1. A booster dose of the Omicron BA.1-adapted bivalentvaccine (30 μg) induced neutralization titers that were approximately3-fold lower than those induced against BA.1. Both Omicron BA.1-adaptedvaccines (e.g., monovalent and bivalent vaccines) were well-tolerated inparticipants who received one or the other Omicron BA.1-adapted vaccine,and demonstrated a favorable safety and tolerability profile similar tothat of BNT162b2 (encoding a SARS-CoV-2 S protein from a Wuhan strain).

Additionally, in a SARS-CoV-2 live virus neutralization assay tested onsera from participants over 56 years of age and older receiving anOmicron-adapted vaccine (e.g., monovalent or bivalent vaccine asdescribed in this Example), sera also neutralized Omicron BA.4/BA.5 withtiters lower than Omicron BA.1.

Example 13: Omicron Breakthrough Infection Drives Cross-VariantNeutralization and Memory B Cell Formation, but to a Lesser ExtentAgainst Omicron BA.4 and BA.5

New Omicron sublineages that harbor further alterations in theSARS-CoV-2 S protein continue to arise, with BA.4 and BA.5 deemed VOCsby the European Centre for Disease Prevention and Control (ECDC) on theMay 12, 2022 (European Centre for Disease Prevention and Control,Epidemiological update: SARS-CoV-2 Omicron sub-lineages BA.4 and BA.5(2022) (available athttps://www.ecdc.europa.eu/en/news-events/epidemiological-update-sarscov-2-omicron-sub-lineages-ba4-and-ba5)).

The present Example 13 is an extension of Example 7, in which the serumsamples collected from BA.1-breakthrough cases as described in Example 7were further analyzed for their neutralization activity against OmicronBA.4 and BA.5 variants.

As described in Example 7, in Omicron-naïve double-vaccinatedindividuals, 50% pseudovirus neutralization (pVN₅₀) geometric meantiters (GMTs) of Beta and Delta VOCs were found to be reduced ascompared to the Wuhan strain, while neutralization of Omicronsublineages BA.1 and BA.2 was virtually undetectable. In this presentExample, FIG. 26(a) shows that neutralization titers of BA.4/5 was alsovirtually undetectable in double-vaccinated, BA.1-breakthrough patients.

As described in Example 7, Omicron-naïve triple-vaccinated individualsexhibited pVN₅₀ GMTs against all tested VOCs that were substantiallyhigher as compared to double-vaccinated individuals. Robustneutralization of Alpha, Beta and Delta variants was observed, whileneutralization of Omicron BA.1 and BA.2 was reduced as compared to Wuhan(GMT 160 and 211 vs 398). As shown in FIG. 26(A) of the present Example,neutralization of Omicron BA.4/5 was further reduced (GMT 74) inOmicron-naïve triple-vaccinating patients, corresponding to a 5-foldlower titer as compared to the Wuhan strain. As shown in FIG. 26(b),Omicron BA.1 breakthrough infection was found to have only a minorboosting effect on neutralization of BA.4/5. In double-vaccinatedpatients, pVN₅₀ GMTs against Omicron BA.4/5 were significantly belowthose against Wuhan (GMT 135 vs. 740). A similar pattern was observedwith BA.1 convalescent and control sera from triple-vaccinatedindividuals. As noted in Example 7, BA.1 convalescent sera exhibitedhigh pVN₅₀ GMTs against previous SARS-CoV-2 VOCs, including Beta (1182),Omicron BA.1 (1029), and Omicron BA.2 (836), which were close to titersagainst the Wuhan reference (1182). In contrast, as shown in FIG. 26(b),neutralization of BA.4/5 in triple-vaccinated individuals with abreakthrough infection of BA.1 was significantly reduced as compared tothe Wuhan strain, with pVN₅₀ GMTs of 197, 6-fold lower than against theWuhan strain.

Of note, in all cohorts, neutralizing titers against BA.4/5 were closerto the low level observed against the phylogenetically more distantSARS-CoV-1 pseudovirus than that seen against Wuhan. Comparing theratios of SARS-CoV-2 VOC and SARS-CoV-1 pVN₅₀ GMTs normalized againstWuhan (FIG. 26(c)), it is remarkable that breakthrough infection withOmicron BA.1 does not lead to more efficient cross-neutralization ofOmicron BA.4/5 in double-vaccinated and triple-vaccinated individuals.In aggregate, these data demonstrate that Omicron BA.1 breakthroughinfection of vaccine-experienced individuals mediates broadlyneutralizing activity against BA.1, BA.2 and several previous SARS-CoV-2variants, but not for BA.4/5.

As shown in FIG. 27 , similar results were found for patients previouslyadministered a non-BNT162b2 vaccine and who had a BA.1 breakthroughinfection.

As described in Example 7, Omicron BA.1 breakthrough infection inBNT162b2-vaccinated individuals was found to produce strong neutralizingactivity against Omicron BA.1, BA.2 and previous SARS-CoV-2 VOCs,primarily by expanding B_(MEM) cells against epitopes shared broadlyacross the different SARS-CoV-2 strains. These data demonstrate that avaccination-imprinted B_(MEM) cell pool has sufficient plasticity to beremodeled by exposure to a heterologous SARS-CoV-2 S protein. Whileselective amplification of B_(MEM) cells recognizing shared epitopesallows for effective neutralization of most variants that evadepreviously established immunity, susceptibility to escape by variantsthat acquire alterations at hitherto conserved sites may be heightened.The significantly reduced neutralizing activity against the OmicronBA.4/5 pseudovirus, which harbors the additional alterations L452R andF486V in the RBD, supports a mechanism of immune evasion by loss of thefew remaining conserved epitopes.

Discussion

Surprisingly, and contrary to the results observed in Example 7,neutralization of Omicron sublineages BA.4 and BA.5 was not enhanced inBA.1-breakthrough patients, with titers instead comparable to thoseagainst the phylogenetically more distant SARS-CoV-1. While the presentExample focused on individuals vaccinated with the BNT162b2 mRNAvaccine, in individuals vaccinated with CoronaVac (a whole, inactivatedvirus vaccine developed by Sinovac Biotech), similar observations haverecently been reported, suggesting that Omicron BA.4/5 can bypass BA.1infection-mediated boosting of humoral immunity (Y. Cao et al.,BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicroninfection, bioRxiv: the preprint server for biology (2022)).

The present disclosure provides insights into how immunity againstmultiple variants is achieved in BA.1 breakthrough cases, why OmicronBA.4 and BA.5 sublineages can partially escape neutralization, andprovides vaccination protocols and technologies to enhance protectionacross coronavirus strains and lineages, specifically including acrossOmicron lineages (e.g., including BA.4 and/or BA.5). Without wishing tobe bound by any particular theory, the present disclosure proposes thatinitial exposure to the Wuhan strain S protein may shape formation ofB_(MEM) cells and imprint against novel B_(MEM) cell responsesrecognizing epitopes distinctive for the Omicron BA.1 variant.

Omicron BA.1 breakthrough infection in BNT162b2-vaccinated individualsprimarily expands a broad B_(MEM) cell repertoire against conservedSARS-CoV-2 S protein and RBD epitopes, rather than inducing strictlyOmicron BA.1-specific B_(MEM) cells. As compared to the immune responseinduced by a homologous vaccine booster, an Omicron BA.1 breakthroughinfection leads to a more substantial increase in antibodyneutralization titers against Omicron and a robust cross-neutralizationof many SARS CoV-2 variants.

As noted in Example 7, one potential explanation for the broadneutralization elicited by a BA.1 breakthrough infection is theinduction of broadly neutralizing antibodies. Sera from OmicronBA.1-convalescent vaccinated individuals was found to neutralizeSARS-CoV-2 Omicron BA.4/5 and SARS-CoV-1 to a far lesser extent thanprevious SARS-CoV-2 VOCs including BA.1 and BA.2. This finding indicatesthat Omicron BA.1 infection in vaccinated individuals stimulates B_(MEM)cells that produce neutralizing antibodies against S protein epitopesconserved in the SARS-CoV-2 variants up to and including Omicron BA.2,but that have mostly been lost in BA.4/5 and are for the most part notshared by SARS-CoV-1.

The greater antigenic distance of the Omicron BA.1 S protein fromearlier SARS-CoV-2 strains may promote targeting of conservedsubdominant neutralizing epitopes as recently described to be located,e.g., in cryptic sites within a portion of the RBD distinct from thereceptor-binding motif (Li, Tingting, et al. “Cross-neutralizingantibodies bind a SARS-CoV-2 cryptic site and resist circulatingvariants,” Nature communications 12.1 (2021): 1-12, and Yuan, Meng, etal. “A highly conserved cryptic epitope in the receptor binding domainsof SARS-CoV-2 and SARS-CoV” Science 368.6491 (2020): 630-633) or in themembrane proximal S glycoprotein subunit designated S2 (Pinto, Dora, etal. “Broad betacoronavirus neutralization by a stem helix-specific humanantibody.” Science 373.6559 (2021): 1109-1116. Li, Wenwei, et al.“Structural basis and mode of action for two broadly neutralizingantibodies against SARS-CoV-2 emerging variants of concern.” Cellreports 38.2 (2022): 110210; Hurlburt, Nicholas K., et al. “Structuraldefinition of a pan-sarbecovirus neutralizing epitope on the spike S2subunit.” Communications biology 5.1 (2022): 1-13).

As noted in Example 7, Omicron BA.1-infected individuals appear to havea significantly higher RBD/S protein-specific B_(MEM) cell ratio ascompared to vaccinated Omicron-naïve individuals. Omicron BA.1 carriesmultiple S protein alterations in key neutralizing antibody bindingsites of the NTD (such as del69/70 and del143-145) that dramaticallyreduce the targeting surface for memory B cell responses in this region.Although the Omicron BA.1 RBD harbors multiple alterations, someneutralizing antibody binding sites are unaffected (20). An expansion ofB_(MEM) cells that produce neutralizing antibodies against RBD epitopesthat are not altered in Omicron BA.1, such as those at position L452 asindicated in the present Example, could help to rapidly restoreneutralization of the BA.1 and BA.2 variants. Importantly, the strongneutralization of Omicron BA.1 and BA.2 should not mask the fact thatthe neutralizing B_(MEM) immune response in Omicron BA.1 convalescentvaccinated individuals is driven by a smaller number of epitopes. Thesignificantly reduced neutralizing activity against the Omicron BA.4/5pseudovirus, which harbors the additional alterations L452R and F486V inthe RBD, demonstrates the mechanism of immune evasion by loss of the fewremaining conserved epitopes. Meanwhile, further sublineages with L452alterations (e.g., BA.2.12.1) are being reported to evade humoralimmunity elicited by BA.1 breakthrough infection (Y. Cao et al., citedabove).

The present disclosure proposes that immunity in the early stages ofOmicron BA.1 infection in vaccinated individuals may be based onrecognition of conserved epitopes, and narrowly focused on a smallnumber of neutralizing sites that are not altered in Omicron BA.1 andBA.2. Such a narrow immune response bears a high risk that those fewepitopes may be lost by acquisition of further alterations in the courseof the on-going evolution of Omicron and may result in immune escape, asexperienced with sublineages BA.2.12.1, BA.4 and BA.5 (Y. Cao et al.,cited above, and K. Khan et al., Omicron sub-lineages BA.4/BA.5 escapeBA.1 infection elicited neutralizing immunity (2022)). Importantly,Omicron BA.1 breakthrough infection does not appear to reduce theoverall spectrum of (Wuhan) S glycoprotein-specific memory B cells, asmemory B cells that do not recognize Omicron BA.1 S remain detectable inblood at similar frequencies. Wuhan-specific (non-Omicron BA.1 reactive)B_(MEM) cells were consistently detected in Omicron BA.1 breakthroughinfected individuals at levels similar to those in Omicron-naïvedouble-/triple-vaccinated individuals. Without wishing to be bound byany particular theory, the present disclosure notes that these findingsmay reflect an increase of the total B_(MEM) cell repertoire byselective amplification of B_(MEM) cells that recognize shared epitopes.

The present Example, among other things, provides insights that it maybe more beneficial for a subject who has been infected or administeredat least one dose (including, e.g., at least two, at least three doses)of vaccine(s) adapted to a Wuhan strain (e.g., but not limited to aprotein based vaccine or RNA-based vaccines such as BNT162b2, ModernamRNA-1273) to receive at least one dose of a vaccine (e.g., a protein orRNA-based vaccine) adapted to a strain that is not an Omicron BA.1. Insome embodiments, a vaccine that is adapted to a strain that is not anOmicron BA.1 can be or comprise a vaccine that is adapted to OmicronBA.4 and/or Omicron BA.5. The present Example, among other things, alsoprovides insights that vaccine-naïve subjects without prior SARS-CoV-2infection may be desirable to be administered a combination of vaccines,which comprises at least one dose of a vaccine adapted to a Wuhan strain(e.g., RNA vaccine such as in some embodiment BNT162b2) and at least onedose of a vaccine adapted to a strain that is not an Omicron BA.1. Insome embodiments, such vaccines in a combination may be administered atdifferent times, for example, in some embodiments as primary dosesand/or booster doses administered apart by a pre-determined period oftime (e.g., according to certain dosing regimens as described herein).In some embodiments, such vaccines in a combination may be administeredas a single multivalent vaccine.

Materials and Methods

Serum samples, neutralization assays, and all other experimentsdescribed in the present Example were performed as in Example 7. TheBA.4/5 VSV-SARS-CoV-2 S variant pseudovirus generation A recombinantreplication-deficient vesicular stomatitis virus (VSV) vector thatencodes green fluorescent protein (GFP) and luciferase instead of theVSV-glycoprotein (VSV-G) was pseudotyped comprised a SARS-CoV-2 Sprotein comprising the following mutations relative to the Wuhan strain:T19I, Δ 24-26, A27S, Δ69/70, G142D, V213G, G339D, S371F, S373P, S375F,T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V,Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H,N969K).

Example 14. Omicron BA.2 Breakthrough Infection of VaccinatedIndividuals Induces Broad Cross Neutralization Against Omicron BA.1,BA.2 and Other VOCs, Including BA.4 and BA.5

The present Example shows that a BA.2 Omicron breakthrough infection inindividuals triple-vaccinated with BNT162b2 surprisingly drives superiorcross variant neutralization as compared to a BA.1-breakthroughinfection in individuals triple-vaccinated with BN162b2, includingimproved production of neutralizing antibodies against a BA.4/5 Omicronvariant. Thus, among other things, the present disclosure demonstratesfeasibility of defining immunologically synergistic categories ofcoronavirus strains and/or sequences (e.g., spike protein sequences).

In some embodiments of improved coronavirus vaccination strategiesprovided by the present disclosure, a subject is exposed to each of atleast two different such synergistic categories. In some embodiments, asubject is, has been or becomes infected with a virus of a firstcategory, and receives at least one dose of a vaccine of a secondcategory, characterized by immunologic synergy with the first category.Alternatively or additionally, in some embodiments, a subject receivesor has received doses of first and second vaccines of such first andsecond categories. In some embodiments, vaccines of different categoriesmay be separately administered (e.g., at different points in time and/orto different sites on a subject). In some embodiments, vaccines ofdifferent categories may be administered together (e.g., atsubstantially the same time and/or to approximately or exactly the samesite and/or in a single composition).

As compared to Omicron BA.1 breakthrough infection of vaccinatedindividuals, which induces lower neutralization against OmicronBA.4/BA.5 relative to neutralization against other SARS-CoV-2 variants(including, e.g., Wuhan-Hu-1 strain, alpha variant, beta variant, deltavariant, Omicron BA.1, Omicron BA.2, and Omicron BA.2.11.2), the presentExample shows that a BA.2 Omicron breakthrough infection in individualsvaccinated with BNT162b2 surprisingly drives superior cross variantneutralization, including improved production of neutralizing antibodiesagainst a BA.4/5 Omicron variant. Thus, in some embodiments, the presentdisclosure, among other things, demonstrates that SARS-CoV-2 strainsand/or variants can be grouped into at least two different categoriessuch that a subject who is exposed to a SARS-CoV-2 strain and/or variantfrom each of such two different categories can benefit fromimmunologically synergistic protection conferred by such two differentcategories. In some embodiments, a first category of SARS-CoV-2strains/variants comprises: Wuhan-Hu-1 strain, alpha variant, betavariant, delta variant, Omicron BA.1, and subvariants derived fromaforementioned strains and/or variants; while a second categorycomprises Omicron BA.2, Omicron BA.2.12.1, Omicron BA.4/BA.5, andsubvariants derived from aforementioned strains and/or variants. Thus,in some embodiments, the present disclosure, among other things, provideinsights that a combination of at least one dose (including, e.g., atleast 1, at least 2, at least 3, at least 4, or more) of a first vaccine(e.g., an mRNA vaccine as described herein that encodes a spike proteinpolypeptide) that comprises or delivers a SARS-CoV-2 spike proteinpolypeptide with a sequence characteristic of a first category asdescribed above, and at least one dose (including, e.g., at least 1, atleast 2, at least 3, at least 4, or more) of a second vaccine (e.g., anmRNA vaccine described herein that encodes a spike protein polypeptide)that comprises or delivers a SARS-CoV-2 spike protein polypeptide with asequence characteristic of a second category as described above cansynergistically provide superior cross variant neutralization, includingenhanced production of neutralizing antibodies toward a BA.4/5 Omicronvariant. In some embodiments, the present disclosure specificallyteaches surprising efficacy of administering at least one dose of avaccine (e.g., an mRNA vaccine that encodes a spike protein polypeptideas described herein) that comprises or delivers a SARS-CoV-2 spikeprotein polypeptide with sequences characteristic of a BA.2 Omicronvariant to subjects who have received at least one (e.g., 2, 3, or more)doses of a vaccine (e.g., vaccine that encodes a spike proteinpolypeptide as described herein) that comprises or delivers a SARS-CoV-2spike protein polypeptide with sequences characteristic of a Wuhan-Hu-1strain).

Background

Emergence of the SARS-CoV-2 Omicron variant of concern (VOC) in November2021 (Ref. 1) can be considered a turning point in the COVID-19pandemic, owing to its ability to substantially escape previouslyestablished immunity. Omicron BA.1, which displaced Delta within weeksas the predominant circulating VOC, had acquired significant alterationsin the receptor binding domain (RBD) and N-terminal domain (NTD) (Ref.2). These changes resulted in a loss of many epitopes recognized byneutralizing antibodies (Refs. 3-4) and drastically impaired humoralimmunity induced by vaccines based on the ancestral Wuhan strain orexposure to the ancestral strain or previous variants (Refs. 5-7). BA.1was subsequently displaced by the BA.2 variant, which in turn gave riseto further sub-lineages. BA.4 and BA.5, which are derived from BA.2, arecurrently becoming the dominant variants in many countries across theglobe with multiple studies suggesting a significant change in antigenicproperties compared to BA.2, and especially compared to BA.1 (Refs.8-9). As BA.4 and BA.5 share an identical S glycoprotein sequence, theyare referred herein as BA.4/5. While many of the amino acid changes inthe RBD are shared between Omicron sub-lineages, alterations within theNTD of BA.2-derived sub-lineages including BA.4/5 are mostly distinctfrom those found in BA.1 (FIG. 33 ).

A vast majority worldwide have been immunized with the vaccines adaptedWuhan strain, including, e.g., mRNA vaccines such as BNT162b2 andmRNA-1273 (Ref. 10), which have thus substantially shaped SARS-CoV-2population immunity. However, emergence of the immune escape variantOmicron BA.1 led to a steep increase in the occurrence of breakthroughinfections in vaccinated individuals. It has been reported thatSARS-CoV-2 variant breakthrough infection can reshape humoral immunity,thereby modulating neutralizing antibody titers against other variants(Refs. 8, 11, 12). However, as previously reported, BA.1 breakthroughinfection may not provide strong immunity against Omicron BA.4/5.

Certain Findings

In order to determine if BA.2 breakthrough infection would refocusimmunity against Omicron BA.2 and BA.2-derived sub-lineages such asBA.4/5, the magnitude and breadth of the neutralizing antibody responsewas studied in samples from individuals who had received a triplevaccination scheme with mRNA vaccines (BNT162b2/mRNA-1273) andsubsequently experienced SARS-CoV-2 breakthrough infections betweenMarch and May 2022, during which period the BA.2 lineage was dominant inGermany (All Vax+Omi BA.2). Such findings have important implicationsfor ongoing efforts of vaccine design, as containment of the COVID-19pandemic requires the generation of durable and sufficiently broadimmunity to provide protection against current and future variants ofSARS-CoV-2.

Two reference cohorts were generated from data previously published inQuandt et al. (Ref. 12), comprising (i) individuals triple-vaccinatedwith BNT162b2 without a prior or breakthrough SARS-CoV-2 infection atthe time of sample collection (BNT162b2³) and (ii) individuals who weretriple-vaccinated with mRNA vaccines with subsequentbreakthrough-infection during a period of Omicron BA.1 dominance (AllVax+Omi BA.1).

Breakthrough infection with the SARS-CoV-2 Omicron BA.1 and BA.2occurred at a median of approximately 4 months or 3 weeks, respectively,after triple-vaccination with an mRNA-based COVID-19 vaccine (BNT162b2,mRNA-1273, or heterologous regimens comprising both vaccines; allVax+Omi BA.1, all Vax+Omi BA.2) (FIG. 29 ). Immune sera used tocharacterize serum neutralizing activity were collected at a median 28days post-vaccination for the BNT162b2³ cohort, 43 days post-BA.1breakthrough for the All Vax+Omi BA.1 cohort, and 39 days post BA.2breakthrough infection for the All Vax+Omi BA.2 cohort. Median ages ofthe cohorts were similar (32-38 years). The BA.2.12.1 neutralizationdata was generated from serum samples from cohorts BNT162b³ and AllVax+Om BA. 1 for this study.

To evaluate the neutralizing activity of immune sera, a pseudovirusneutralization test (pVNT), for example, as described in Refs. 13, 14,were used. Pseudoviruses bearing the S glycoproteins of SARS-CoV-2Wuhan, Alpha, Beta, Delta, Omicron BA.1, BA.2, BA.2.12.1, as well as therecently emerged Omicron sub-lineages BA.4 and BA.5 were applied toassess neutralization breadth. As BA.4 and BA.5 share an identical Sglycoprotein sequence, including key alterations L452R and F486V, theyare referred herein as BA.4/5. In addition, SARS-CoV (herein referred toas SARS-CoV-1; Ref. 15) was assayed to detect potential pan-Sarbecovirusneutralizing activity.

As reported previously in Ref. 12, 50% pseudovirus neutralization(pVN₅₀) geometric mean titers (GMTs) against Omicron BA.1 and BA.2 ofimmune sera from SARS-CoV-2 naïve triple-vaccinated individuals wereconsiderably reduced compared to the Wuhan strain (GMT 160 and 221versus 398). Neutralizing activity against BA.2.12.1 and BA.4/5 was evenfurther reduced (GMTs 111 and 74), corresponding to a 5.4-fold lowertiter for BA.4/5 as compared to the Wuhan strain (FIG. 30(A)).

Omicron BA.2 breakthrough infection markedly increased pVN₅₀ GMTsagainst BA.2 and BA.2.12.1 compared to SARS-CoV-2-naïvetriple-vaccinated immune sera, such that neutralization of BA.2 afterbreakthrough infection was comparable to the Wuhan strain (FIG. 31(B-C)). Similarly, BA.1 breakthrough infection conferred robustneutralizing activity against BA.1 (FIG. 30(B), FIG. 31(A). Importantly,while pVN₅₀ GMTs against BA.4/5 in BA.2 convalescent sera were lowerthan against the Wuhan strain (GMTs 391 versus 922, i.e., 2.4-foldreduction), this reduction was still less than that observed in theOmicron-naïve BNT162b2³ cohort, whose sera showed a 5.4-fold reductionof BA.4/5 neutralizing activity (FIG. 31(C)). By contrast, pVN₅₀ GMTsagainst BA.4/5 and Wuhan after BA.1 breakthrough infection were 266 and1327, respectively (i.e., 5-fold reduction; FIG. 30(B)). Hence, OmicronBA.1 breakthrough infection of triple-vaccinated individuals did notlead to more efficient cross-neutralization of Omicron BA.4/5 ascompared with triple-vaccinated Omicron-naïve individuals. In bothcohorts, neutralizing titers against BA.4/5 were closer to the low levelobserved against the phylogenetically more distant SARS-CoV-1 than thatseen against Wuhan (FIG. 30 ). Of note, the pVN₅₀ GMTs against the Wuhanstrain after BA.1 breakthrough infection were slightly higher than thoseobserved for BA.2 breakthrough infection (GMTs 1327 versus 922), which,without wishing to be bound by a particular theory, may relate to thelonger interval between the third vaccination and the infection (median22 days for BA.1 versus 127.5 days for BA.2) (FIG. 31 ). A separateanalysis was conducted including only individuals triple-vaccinated withBNT162b2 (with BA.2 or BA.1 breakthrough infections, or Omicron-naïve).In these analyses similar observations regarding BA.4/5 neutralizingactivities were made: pVN₅₀ GMTs against BA.4/5 in BA.2 convalescentsera were 2.4-fold lower than against the Wuhan strain, whereas thereduction was 6-fold after BA.1 breakthrough infections (FIG. 31 ).While relative neutralization of BA.2 and BA.2.12.1 was comparable inBA.2 and BA.1 convalescent sera, neutralizing activity against thesevariants remained slightly above that seen in Omicron-naïve sera.

Immune sera from triple-vaccinated Omicron naïve individuals had broadneutralizing activity against ancestral SARS-CoV-2 VOCs. Neutralizingactivity against Beta was slightly higher in BA.1 convalescent sera,whereas neutralization of Alpha and Delta was not affected by BA.1 orBA.2 breakthrough infections (FIG. 31(C)).

In aggregate, these data demonstrate that Omicron BA.2 breakthroughinfections of vaccine-experienced individuals mediate broadlyneutralizing activity against BA.1, BA.2, BA.2.12.1 and severalancestral SARS-CoV-2 variants. Moreover, neutralizing activity againstBA.4/5, while lower than against the Wuhan reference, is provided to alarger extent than in BA.1 convalescent sera.

Recent studies have demonstrated that Omicron BA.1 breakthroughinfection in individuals vaccinated with an mRNA vaccine (BNT162b2 ormRNA-1273) boosts serum neutralizing titers not only against theancestral Wuhan strain, but also against VOCs including BA.2 (Refs. 8,11, 12). This effect was seen in triple-vaccinated individuals but wasparticularly evident in double-vaccinated individuals, whose seracontain little to no neutralizing activity against BA.2. However, BA.1breakthrough infection did not induce strong neutralizing activityagainst BA.4/5, VOCs that are currently establishing dominanceworldwide. Without wishing to be bound by a particular theory, thisimmune escape has been attributed to the amplification and/or recall ofpre-existing neutralizing antibody responses that recognize epitopesabsent in the Omicron sub-lineages BA.2.12.1, BA.4, and BA.5.

The present Example, among other things, provide insights that BA.2breakthrough infections trigger recall responses which mediate enhancedneutralization of the BA.2-derived sub-lineages, including BA.4/5,indicating that higher S protein sequence similarity among BA.2,BA.2.12.1, and BA.4/5 drives more efficient cross-neutralizationcompared to breakthrough infections with the more distant BA.1 variant.Notwithstanding the importance of vaccination with currently approvedWuhan-derived vaccines such as BNT162b2 that offer effective protectionfrom severe disease by current VOCs including Omicron BA.1 and BA.2, thepresent findings of broadly cross-neutralizing activity against currentVOCs including BA.4/5 after BA.2 breakthrough infection providesinsights, among other things, that a combination of a vaccine adapted toWuhan strain sequence or a variant sequence from the sameimmunologically-related category as discussed above (e.g., alpha strain,beta strain, delta strain, Omicron BA.1) and a vaccine adapted to theBA.2 variant sequence or a variant sequence from the sameimmunologically-related category as discussed above (e.g., OmicronBA.2.12.1, Omicron BA.4/BA.5) can provide enhanced cross-neutralizationactivity against variants from two different categories. In someembodiments, the present Example provides evidence that supportsimplementation of licensure procedures modelled on that of seasonal fluvaccines that use the latest epidemiological data to select for COVID-19vaccine strains. In some embodiments, the present Example furtherprovides evidence that supports establishment of rapid strain selectionfor seasonal updates of COVID-19 vaccines, similar to the selectionprocess practiced by the World Health Organization (WHO) GlobalInfluenza Surveillance and Response System (GISRS), and/or agreement onaccelerated approval pathways based on surrogate immunogenicityendpoints.

Neutralization titers from subjects vaccinated against SARS-CoV-2 andwho have had a BA.1 or a BA.2 breakthrough infection are shown in FIGS.31(A) and (B), respectively, and GMRs for both groups of subjects areshown in FIG. 31(C). As shown in FIGS. 31(A) and (B), sera from subjectspreviously vaccinated against SARS-CoV-2, and who had a breakthroughinfection with either BA.1 or BA.2, were found to have significantneutralization titers against pseudovirus comprising a SARS-CoV-2 Sprotein of a Wuhan strain, an Alpha variant, a Beta variant, a DeltaVariant, and an Omicron BA.1 variant. As noted previously,neutralization titers against BA.2 are somewhat lower in sera from BA.1breakthrough patients (GMT of 875 for BA.2 vs 1327 for Wuhan strain) andare lower still against BA.2.12.1 and BA.4/5 (GMTs of 584 and 266,respectively as compared to 1327 for Wuhan). BA.2 breakthrough patientsshow similar neutralization responses as BA.1 breakthrough patientsagainst a SARS-CoV-2 Wuhan strain, Alpha variant, Beta variant, andDelta variant. The neutralization response against Omicron BA.1 issomewhat higher in BA.1-breakthrough patients than in BA.2-breakthroughpatients (GMR of 0.76 as compared to 0.60), while neutralization titersagainst Omicron BA.2 are higher in BA.2-breakthrough patients than inBA.1-breakthrough patients (GMR of 0.94 vs 0.66). Surprisingly, however,neutralization responses against BA.4/5 are significantly higher inBA.2-breakthrough patients (GMR of 0.39 in BA.2 breakthrough patients,as compared to a GMR of 0.2 in BA.1 breakthrough subjects). The presentdisclosure therefore documents that a broader immune response can beelicited by a BA.2-breakthrough infection as compared to a BA.1breakthrough infection in subject vaccinated against SARS-CoV-2, andteaches that administering a booster vaccine comprising RNA encoding anS protein comprising mutations characteristic of a BA.2 Omicron variantcan achieve surprising and unexpected benefits.

Furthermore, the present disclosure provides an insight that, givensimilarities among S protein sequences of BA.2 and BA.4/5 variants,combining vaccination doses that comprise or deliver BA.4 and/or BA.5variant spike sequences with those of that comprise or deliver Wuhanspike sequences may also achieve particularly broad immunization (i.e.,synergistic immunization as described herein).

In some embodiments, these findings suggest that synergistic categoriesof coronavirus strain and/or variant sequences (e.g., SARS-CoV-2 strainand/or variant sequences) can be defined, for example, in someembodiments based on shared amino acid alterations in S glycoprotein ofcoronavirus strain and/or variant sequences. For example, while many ofthe amino acid changes in the RBD of S protein are shared betweenOmicron sub-lineages (e.g., BA.1, BA.2, BA.2.12.1, and BA.4/5),alterations within the NTD of BA.2 and BA.2-derived sub-lineagesincluding BA.4/5 are mostly distinct from those found in BA.1.Therefore, in some embodiments, synergistic categories of coronavirusstrain and/or variant sequences (e.g., SARS-CoV-2 strain and/or variantsequences) can be defined based on the degree of shared amino acidmutations present with the NTD of a S protein. For example, in someembodiments where two SARS-CoV-2 strain and/or variant sequences shareat least 50% (including, e.g., at least 60%, at least 70%, at least 80%,at least 90%, or more) of the amino acid mutations present in the NTD ofa S protein, both SARS-CoV-2 strain and variant sequences can be groupedinto the same category. In some embodiments where two SARS-CoV-2 strainand/or variant sequences share no more 50% (including, e.g., no morethan 45%, no more than 40%, no more than 30%, or lower) of the aminoacid mutations present in the NTD of a S protein, both SARS-CoV-2 strainand variant sequences can be grouped into different categories. Amongother things, the present findings provide insights that exposingsubjects (e.g., via infection and/or vaccination) to at least twoantigens that are of different synergistic categories (e.g., as shown inthe table below) can produce a more robust immune response (e.g.,broadening the spectrum of cross-neutralization against differentvariants and/or producing an immune response that is less prone toimmune escape).

Category I Category II Wuhan strain Omicron BA.2 Alpha variant OmicronBA.2.11.2 Beta variant Omicron BA.4 Delta variant Omicron BA.5 OmicronBA.1 Sublineages derived from any one of the above Sublineages derivedfrom any one of the above

For example, in some embodiments, vaccine-naïve subjects without priorinfection may be administered a combination of vaccines, at least two ofwhich are each adapted to a SARS-CoV-2 strain of different synergisticcategories (e.g., as described herein). In some embodiments, suchvaccines in a combination may be administered at different times, forexample, in some embodiments as a first dose and a second doseadministered apart by a pre-determined period of time (e.g., accordingto certain dosing regimens as described herein). In some embodiments,such vaccines in a combination may be administered as a multivalentvaccine. In some embodiments, subject infected or vaccinated with aSARS-CoV-2 strain of one category may be administered with a vaccineadapted to a SARS-CoV-2 strain of a different category (e.g., asdescribed herein). In some embodiments, such a vaccine may be apolypeptide-based or RNA-based vaccine.

In some embodiments, a combination of vaccines comprising a vaccine fromCategory I and a BA.4/5 vaccine may provide a particularly superiorimmune response (e.g., an immune response with particularly strongcross-neutralization effects).

Due to the large number of differences between XBB and its variants, insome embodiments, a particularly improved synergistic effect can beproduced by a combination comprising a vaccine of Category I and an XBBvaccine. In some embodiments, a particularly improved synergistic effectcan be produced by a combination comprising a BA.4/5 vaccine and an XBBvaccine.

While the present findings are based on retrospective analyses ofsamples derived from different studies, using relatively small samplessizes and cohorts that are not fully aligned regarding immunizationintervals and demographic characteristics such as age and sex ofindividuals, the present findings provide useful insights for vaccinedesign and vaccination strategies for improving cross-neutralizationagainst a broader spectrum of SARS-COV-2 variants.

In some embodiments, a vaccine can comprise a polypeptide (e.g., anon-natural polypeptide, e.g., a chimeric polypeptide) comprising one ormore mutations that are characteristic of one or more differentSARS-CoV-2 variants, or a nucleic acid (e.g., in some embodiments anRNA) encoding the polypeptide. In some embodiments, a vaccine cancomprise a polypeptide (e.g., a non-natural polypeptide, e.g., achimeric polypeptide) comprising one or more mutations that arecharacteristic of a first SARS-CoV-2 variant and one or more mutationsthat are characteristic of a second SARS-CoV-2 variant, or a nucleicacid (e.g., in some embodiments an RNA) encoding the polypeptide. Insome embodiments, a first SARS-CoV-2 variant can be a SARS-CoV-2strain/variant from Category I of the table above, while a secondSARS-CoV-2 variant can be a SARS-CoV-2 strain/variant from Category IIof the table above. For example, in some embodiments, a vaccine cancomprise a polypeptide that comprises an RBD comprising one or moremutations that are characteristic of a first SARS-CoV-2 variant and anNTD comprising one or more mutations that are characteristic of a secondSARS-CoV-2 variant, or a nucleic acid (e.g., in some embodiments an RNA)encoding the polypeptide.

In some embodiments, a vaccine can comprise a polypeptide that comprisesan RBD comprising one or more mutations characteristic of a BA.1 Omicronvariant and an NTD comprising one or more mutations characteristic of asecond SARS-CoV-2 variant that is not a BA.1 Omicron variant, or anucleic acid (e.g., in some embodiments an RNA) encoding thepolypeptide. In some embodiments, a vaccine can comprise or encode apolypeptide comprising an NTD comprising one or more mutationscharacteristic of a BA.1 Omicron variant and an RBD comprising one ormore mutations characteristic of a second SARS-CoV-2 variant that is nota BA.1 Omicron variant. In some embodiments, a vaccine can comprise apolypeptide that comprises an RBD comprising one or more mutationscharacteristic of a BA.1 Omicron variant and an NTD comprising one ormore mutations characteristic of a BA.2 Omicron variant, or a nucleicacid (e.g., in some embodiments an RNA) encoding the polypeptide. Insome embodiments, a vaccine can comprise a polypeptide that comprises anRBD comprising one or more mutations characteristic of a BA.1 Omicronvariant and an NTD comprising one or more mutations characteristic of aBA.4/5 Omicron variant, or a nucleic acid (e.g., in some embodiments anRNA) encoding the polypeptide.

In some embodiments, a vaccine can comprise a polypeptide that comprisesan NTD comprising one or more mutations characteristic of a BA.1 Omicronvariant and an RBD comprising one or more mutations characteristic of asecond SARS-CoV-2 variant that is not a BA.1 Omicron variant, or anucleic acid (e.g., in some embodiments an RNA) encoding thepolypeptide. In some embodiments, a vaccine can comprise a polypeptidethat comprises an RBD comprising one or more mutations characteristic ofa BA.1 Omicron variant and an NTD comprising one or more mutationscharacteristic of a second SARS-CoV-2 variant that is not a BA.1 Omicronvariant, or a nucleic acid (e.g., in some embodiments an RNA) encodingthe polypeptide. In some embodiments, a vaccine can comprise apolypeptide that comprises an NTD comprising one or more mutationscharacteristic of a BA.1 Omicron variant and an RBD comprising one ormore mutations characteristic of a BA.2 Omicron variant, or a nucleicacid (e.g., in some embodiments an RNA) encoding the polypeptide. Insome embodiments, a vaccine can comprise a polypeptide that comprisesone or more mutations characteristic of an NTD of a BA.1 Omicron variantand an RBD comprising one or more mutations characteristic of a BA.4/5Omicron variant, or a nucleic acid (e.g., in some embodiments an RNA)encoding the polypeptide.

In some embodiments, a vaccine can comprise a polypeptide that comprisesan RBD comprising one or more mutations characteristic of a BA.1 Omicronvariant and an NTD of a Wuhan S protein, or a nucleic acid (e.g., insome embodiments an RNA) encoding the polypeptide. In some embodiments,a vaccine can comprise a polypeptide that comprises an RBD comprisingone or more mutations characteristic of a BA.2 Omicron variant and anNTD of a Wuhan S protein, or a nucleic acid (e.g., in some embodimentsan RNA) encoding the polypeptide. In some embodiments, a vaccine cancomprise a polypeptide that comprises an RBD comprising one or moremutations characteristic of a BA.4/5 Omicron variant and an NTD of aWuhan S protein, or a nucleic acid (e.g., in some embodiments an RNA)encoding the polypeptide.

In some embodiments, a vaccine can comprise a polypeptide that comprisesan NTD comprising one or more mutations characteristic of a BA.1 Omicronvariant and an RBD of a Wuhan S protein, or a nucleic acid (e.g., insome embodiments an RNA) encoding the polypeptide. In some embodiments,a vaccine can comprise a polypeptide that comprises an NTD comprisingone or more mutations characteristic of a BA.2 Omicron variant and anRBD of a Wuhan S protein, or a nucleic acid (e.g., in some embodimentsan RNA) encoding the polypeptide. In some embodiments, a vaccine cancomprise a polypeptide that comprises an NTD comprising one or moremutations characteristic of a BA.4/5 Omicron variant and an RBD of aWuhan S protein, or a nucleic acid (e.g., in some embodiments an RNA)encoding the polypeptide.

Materials and Methods

Recruitment of Participants and Sample Collection

Individuals from the SARS-CoV-2-naïve BNT162b2 triple-vaccinated(BNT162b2³) cohort provided informed consent as part of theirparticipation in the Phase 2 trial BNT162-17 (NCT05004181). Individualswith Omicron BA.1 or BA.2 breakthrough infection (All Vax+Omi BA.1 andAll Vax+Omi BA.2 cohorts) were triple-vaccinated, e.g., with one or moredoses of BNT162b2, Moderna mRNA-1273, AstraZeneca ChAdOx1-S recombinantvaccine, or a combination thereof, and were recruited to provide bloodsamples and clinical data for research. Omicron infections wereconfirmed with variant-specific PCR either between November 2021 andmid-January 2022 (All Vax+Omi BA.1) or between March 2022 and May 2022,at times were sub-lineages BA.1 or BA.2, respectively, were dominant(Ref. 24). The infections of certain participants (e.g., at least 7participants) in this study were further characterized by genomesequencing, and genome sequencing confirmed Omicron BA.1 or BA.2infection.

Participants were free of symptoms at the time of blood collection.Table 26 is a summary of characteristics of vaccinated individualsanalyzed for neutralizing antibody responses. All participants had nodocumented history of SARS-CoV-2 infection prior to vaccination.

TABLE 26 all Vax + all Vax + Omi BA.2 Omi BA.1 BNT16262³ Characteristic(n = 14) (n = 15) (n = 19) Sex, n (%) Male 6 (43) 12 (80) 10 (53) Female8 (57) 3 (20) 9 (47) Age, median 34 (25-57) 32 (23-60) 38 (23-54)(range) Age group at vaccination, n (%) 18-55 yrs 12 (86) 13 (87) 19(100) 56-85 yrs 2 (14) 2 (13) 0 (0) Baseline SARS-CoV-2 status, n (%)Positive 14 (100)† 15 (100)† 0 (0) Negative 0 (0) 0 (0) 19 (100)^(#)Unknown 0 (0) 0 (0) 0 (0) Interval, median (range) Days between 41(15-88) 39 (20-92) ‡ D 1/D 2 Days between 189 (152-249) 189 (156-256)202 (181-266) D 2/D 3 Days until N/A N/A 28 (26-30) serum draw after D 3Days between 127.5 (36-188) 22 (3-112) N/A last dose/ infection Daysuntil 43 (34-99) 43 (25-55) N/A serum draw after infection N/A: notapplicable; n/a, not available; D, Dose; Yrs, Years; n, Number.*Negative SARS-CoV-2 PCR test at the time of enrollment ^(#)No evidenceof prior SARS-CoV-2 infection (based on COVID-19 symptoms/signs andSARS-CoV-2 PCR test) ‡, Participants received the primary 2-dose seriesof BNT162b2 vaccine as part of a governmental vaccination program andthe interval between doses was not recorded †Omicron BA.1 infectionconfirmed at time of recruitment to the research study.

Serum was isolated by centrifugation of drawn blood at 2000×g for 10minutes and cryopreserved until use.

VSV-SARS-CoV-2 S Variant Pseudovirus Generation

A recombinant replication-deficient vesicular stomatitis virus (VSV)vector that encodes green fluorescent protein (GFP) and luciferaseinstead of the VSV-glycoprotein (VSV-G) was pseudotyped with SARS-CoV-1S glycoprotein (UniProt Ref: P59594) and with SARS-CoV-2 S glycoproteinderived from either the Wuhan reference strain (NCBI Ref: 43740568), theAlpha variant (alterations: Δ69/70, Δ144, N501Y, A570D, D614G, P681H,T716I, S982A, D1118H), the Beta variant (alterations: L18F, D80A, D215G,Δ242-244, R246I, K417N, E484K, N501Y, D614G, A701V), the Delta variant(alterations: T19R, G142D, E156G, Δ157/158, K417N, L452R, T478K, D614G,P681R, D950N) the Omicron BA.1 variant (alterations: A67V, Δ69/70, T95I,G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, 5375F,K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y,Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H,N969K, L981F), the Omicron BA.2 variant (alterations: T19I, Δ24-26,A27S, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S,K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G,H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K), the Omicron BA.2.12.1variant (alterations: T19I, Δ24-26, A27S, G142D, V213G, G339D, S371F,S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452Q, S477N, T478K,E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, S704L,N764K, D796Y, Q954H, N969K), or the Omicron BA.4/5 variant (alterations:T19I, Δ24-26, A27S, Δ69/70, G142D, V213G, G339D, S371F, S373P, S375F,T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V,Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H,N969K) according to published pseudotyping protocols (Ref. 49).

A diagram of SARS-CoV-2 S glycoprotein alterations is shown in FIG. 32and a separate alignment of S glycoprotein alterations in Omicronsub-lineages is displayed in FIG. 33 . In brief, HEK293T/17 monolayers(ATCC® CRL-11268™) cultured in Dulbecco's modified Eagle's medium (DMEM)with GlutaMAX™ (Gibco) supplemented with 10% heat-inactivated fetalbovine serum (FBS [Sigma-Aldrich]) (referred to as medium) weretransfected with Sanger sequencing-verified SARS-CoV-1 orvariant-specific SARS-CoV-2 S expression plasmid with Lipofectamine LTX(Life Technologies) following the manufacturer's instructions. At 24hours VSV-G complemented VSVΔG vector. After incubation for 2 hours at37° C. with 7.5% CO2, cells were washed twice with phosphate bufferedsaline (PBS) before medium supplemented with anti-VSV-G antibody (clone8G5F11, Kerafast Inc.) was added to neutralize residualVSV-G-complemented input virus. VSV-SARS-CoV-2-S pseudotype-containingmedium was harvested hours after inoculation, passed through a 0.2 μmfilter (Nalgene) and stored at −80° C. The pseudovirus batches weretitrated on Vero 76 cells (ATCC® CRL-1587™) cultured in medium. Therelative luciferase units induced by a defined volume of a Wuhan Sglycoprotein pseudovirus reference batch previously described in Muik etal., 2021, that corresponds to an infectious titer of 200 transducingunits (TU) per mL, was used as a comparator. Input volumes for theSARS-CoV-2 variant pseudovirus batches were calculated to normalize theinfectious titer based on the relative luciferase units relative to thereference.

Pseudovirus Neutralization Assay

Vero 76 cells were seeded in 96-well white, flat-bottom plates (ThermoScientific) at 40,000 cells/well in medium 4 hours prior to the assayand cultured at 37° C. with 7.5% CO2. Each individual serum was seriallydiluted 2-fold in medium with the first dilution being 1:5(Omicron-naïve triple BNT162b2 vaccinated; dilution range of 1:5 to1:5,120) or 1:30 (triple vaccinated after subsequent Omicron BA.1 orBA.2 breakthrough infection; dilution range of 1:30 to 1:30,720). In thecase of the SARS-CoV-1 pseudovirus assay, the serum of all individualswas initially diluted 1:5 (dilution range of 1:5 to 1:5,120).VSV-SARS-CoV-2-S/VSV-SARS-CoV-1-S particles were diluted in medium toobtain 200 TU in the assay. Serum dilutions were mixed 1:1 withpseudovirus (n=2 technical replicates per serum per pseudovirus) for 30minutes at room temperature before being added to Vero 76 cellmonolayers and incubated at 37° C. with 7.5% CO2 for 24 hours.Supernatants were removed and the cells were lysed with luciferasereagent (Promega). Luminescence was recorded on a CLARIOstar® Plusmicroplate reader (BMG Labtech), and neutralization titers werecalculated as the reciprocal of the highest serum dilution that stillresulted in 50% reduction in luminescence. Results were expressed asgeometric mean titers (GMT) of duplicates. If no neutralization wasobserved, an arbitrary titer value of half of the limit of detection[LOD] was reported.

Statistical Analysis

The statistical method of aggregation used for the analysis of antibodytiters is the geometric mean and for the ratio of SARS-CoV-2 VOC titerand Wuhan titer the geometric mean and the corresponding 95% confidenceinterval. The use of the geometric mean accounts for the non-normaldistribution of antibody titers, which span several orders of magnitude.The Friedman test with Dunn's correction for multiple comparisons wasused to conduct pairwise signed-rank tests of group geometric meanneutralizing antibody titers with a common control group. Allstatistical analyses were performed using GraphPad Prism softwareversion 9.

REFERENCES CITED IN EXAMPLE 14

-   1. WHO Technical Advisory Group on SARS-CoV-2 Virus Evolution    (TAG-VE), Classification of Omicron (B.1.1.529): SARS-CoV-2 Variant    of Concern (2021).-   2. WHO Headquarters (HQ), WHO Health Emergencies Programme,    Enhancing response to Omicron SARS-CoV-2 variant: Technical brief    and priority actions for Member States (2022).-   3. M. Hoffmann et al., The Omicron variant is highly resistant    against antibody-mediated neutralization. Cell. 185, 447-456.e11    (2022), doi:10.1016/j.cell.2021.12.032.-   4. W. Dejnirattisai et al., SARS-CoV-2 Omicron-B.1.1.529 leads to    widespread escape from neutralizing antibody responses. Cell. 185,    467-484.e15 (2022), doi:10.1016/j.cell.2021.12.046.-   5. V. Servellita et al., Neutralizing immunity in vaccine    breakthrough infections from the SARS-CoV-2 Omicron and Delta    variants. Cell. 185, 1539-1548.e5 (2022),    doi:10.1016/j.cell.2022.03.019.-   6. C. Kurhade et al., Neutralization of Omicron BA.1, BA.2, and BA.3    SARS-CoV-2 by 3 doses of BNT162b2 vaccine. Nature communications.    13, 255 (2022), doi:10.1038/s41467-022-30681-1.-   7. Y. Cao et al., Omicron escapes the majority of existing    SARS-CoV-2 neutralizing antibodies. Nature. 602, 657-663 (2022),    doi:10.1038/s41586-021-04385-3.-   8. Y. Cao et al., BA.2.12.1, BA.4 and BA.5 escape antibodies    elicited by Omicron infection. Nature (2022),    doi:10.1038/s41586-022-04980-y.-   9. N. P. Hachmann et al., Neutralization Escape by SARS-CoV-2    Omicron Subvariants BA.2.12.1, BA.4, and BA.5. The New England    journal of medicine (2022), doi:10.1056/NEJMc2206576.-   10. E. Mathieu et al., A global database of COVID-19 vaccinations.    Nature human behaviour. 5, 947-953 (2021),    doi:10.1038/s41562-021-01122-8.-   11. C. I. Kaku et al., Recall of pre-existing cross-reactive B cell    memory following Omicron BA.1 breakthrough infection. Science    immunology, eabq3511 (2022), doi:10.1126/sciimmunol.abq3511.-   12. J. Quandt et al., Omicron BA.1 breakthrough infection drives    cross-variant neutralization and memory B cell formation against    conserved epitopes. Science immunology, eabq2427 (2022),    doi:10.1126/sciimmunol.abq2427.-   13. A. Muik et al., Neutralization of SARS-CoV-2 Omicron by BNT162b2    mRNA vaccine-elicited human sera. Science (New York, N.Y.). 375,    678-680 (2022), doi:10.1126/science.abn7591.-   14. A. Muik et al., Neutralization of SARS-CoV-2 lineage B.1.1.7    pseudovirus by BNT162b2 vaccine-elicited human sera. Science (New    York, N.Y.). 371, 1152-1153 (2021), doi:10.1126/science.abg6105.-   15. C.-W. Tan et al., Pan-Sarbecovirus Neutralizing Antibodies in    BNT162b2-Immunized SARS-CoV-1 Survivors. The New England journal of    medicine. 385, 1401-1406 (2021), doi:10.1056/NEJMoa2108453.

Example 15: Further Updates on Immune Responses Elicited by VaccinesEncoding a SARS-CoV-2 S Protein from an Omicron Variant

Following the experiment described in Example 8, further subjects wereenrolled in a clinical trial investigating RNA vaccines encoding aSARS-CoV-2 S protein comprising one or more mutations characteristic ofa BA.1 Omicron variant. In the present Example, subjects (18 to 55 yearsof age with or without evidence of prior infection) were administered a2nd booster (4th dose) of either 30 ug of RNA encoding a SARS-CoV-2 Sprotein of a Wuhan strain (in the present Example, BNT162b2) or 30 μg ofRNA encoding an SARS-CoV-2 S protein having one or more mutationscharacteristic of an Omicron variant (in the present example, BNT162b2OMI, which encodes a SARS-CoV-2 S protein having mutationscharacteristic of a BA.1 Omicron variant, comprises SEQ ID NOs: 50 and51, and encodes an amino acid of SEQ ID NO: 49).

In a primary immunogenicity analysis of participants without evidence ofprior infection, BNT162b2 OMI (N=132) elicited a superior neutralizingantibody response to the BA.1 Omicron SARS-CoV-2 virus compared toBNT162b2 (N=141). The BNT162b2 OMI GMT against BA.1 Omicron was 1929(CI: 1632, 2281) compared to a BNT162b2 GMT of 1100 (CI: 932, 1297); GMTratio 1.75 (95% CI: 1.39, 2.22).

Compared to BNT162b2, BNT162b2 OMI elicited a similar neutralizingantibody response to a Wuhan strain of SARS-CoV-2. BNT162b2 OMI GMT was11997 (CI: 10554, 13638) compared to a BNT162b2 GMT of 12009 (CI: 10744,13425).

The data suggests that an Omicron monovalent vaccine as the 2nd boostervaccination (4th dose) improves a neutralizing antibody response to BA.1Omicron compared to an RNA vaccine encoding an S protein of a Wuhanstrain, and does not negatively affect a neutralizing antibody responseto a Wuhan strain of SARS-CoV-2.

Example 16. Immunological Impact of VOC Vaccination

The present Example describes immunological impacts of administration ofBNT162b2 vaccines encoding spike proteins from certain variants ofconcern (“VOC”). In particular, the present Example describesimmunological impacts of administration of a “booster dose” to subjects(in this Example, mice) who have received two doses (i.e., according toan established model immunization protocol) of the “original” BNT162b2vaccine (i.e., encoding the Wuhan spike protein, as described herein).

FIG. 34 presents the immunization protocol utilized in the presentExample. Specifically, BALB/c mice were immunized twice (1 ug each dose)with BNT 162b2, and then at a later time point with a BNT162b2/VOC (1 ugeach dose). Immunization occurred up to 3 or 4 times. Animals were bledregularly to analyze antibody immune response by ELISA and pseudovirusneutralization assay. At the end of the trial, animals were euthanizedand T cell response in the spleen was analyzed.

Boosting was performed with: (a) the original BNT162b2 (“BNT162b2”); (b)BNT162b2 OMI BA.1 (“OMI BA.1”); (c) BNT162b2 OMI BA.4/5 (“OMI BA.4/5”);(d) BNT162b2+OMI BA.1 (0.5 g each) (e) BNT162b2+OMI BA.4/5 (0.5 ugeach); (f) OMI BA.1+OMI BA.4/5 (0.5 ug each); and (g) BNT162b2+OMIBA.1+OMI BA.4/5 (0.33 ug each).

Omicron variants BA.4 and BA.5 were first reported in circulation inJanuary 2022, and were becoming dominant variants by June 2022. Both ofthese lineages contain the amino-acid substitutions F486V, and R493Q.Preliminary studies suggest a significant change in antigenic propertiesof BA.4 and BA.5 compared to BA.1 and BA.2, especially compared to BA.1.Additionally, as increasing trends in BA.5 variant proportions areobserved in particular locations (e.g., in Portugal), COVID-19 casenumbers and test positivity rate have also increased. The presentdisclosure proposes that BA.4/5 (which, given their common spike proteinmutations, are considered together in the present Example) couldrepresent escape VOC. The present disclosure demonstrates particularbenefits of dosing regimens (e.g., as described herein and specificallyas exemplified in the present Example) that include one or more doses ofa vaccine that comprises or delivers (e.g., via expression of anadministered RNA) a spike protein that includes relevant BA.4/5sequences (e.g., amino acid substitutions). FIGS. 35 and 36 presentbaseline (determined at day 104, pre-boost) geometric mean titers(“GMT”s) relative to various SARS-CoV-2 strains, as indicated. As can beseen, baseline immunization of the different mouse cohorts wascomparable. Specifically, group GMTs per pseudovirus were consistentlyin the same ballpark between cohorts; no difference greater than about2-fold was observed. Consistent with observations made in humanpopulations, as noted above, neutralizing GMTs against the Wuhan strainwere considerably higher (GMT of up to 3,044) as compared to thoseagainst VOCs. Overall, the order of GMTs wasWuhan>BA.1≅BA.2>BA.2.12.1>BA.4/5.

FIG. 37 shows baseline (determined at day 104, pre-boost)cross-neutralization analysis and demonstrates that baselineimmunization of cohorts with respect to cross-neutralization capacity iscomparable. Specifically, at baseline, calculated variant/Wuhan ref GMTratios indicated that cross-neutralization capacity was quite comparablebetween cohorts (only one outlier in the BNT162b2 monovalent group re.BA.1 neutralization was observed). Again consistent with observations ofhuman populations, BA.1=BA.2>BA.2.12.1>BA.4/5 FIGS. 38-40 present dataobtained seven days post-boost and document remarkable effectiveness ofBA.4/5, and in particular of monovalent BA.4/5, in achieving significantgeometric mean fold increase of GMTs (FIGS. 38 and 39 ) and effectivecross-neutralization (FIG. 40 ). As can be seen, BNT162b2 boosterimmunization resulted in a comparable titer increase against all VOCs(3.9-7.1 fold), whereas monovalent BA.1 and BA.4/5 boosters resulted ina considerably stronger increase in the homologous VOC titer (16.8-foldfor BA.1, 67.3-fold for BA.4/5).

The monovalent BA.4/5 booster was the most effective in driving titerincreases across the pseudovirus panel tested. Bivalent boosters showeda similar but attenuated trend compared to the monovalent VOC boosters;amongst bivalent boosters the b2+BA.4/5 combination was most effectivein driving broad cross-neutralization. The trivalent booster(b2+BA.1+BA.4/5) was superior to the bivalent boosters and gaveintermediate immunization between the bivalent b2+BA.4/5 and monovalentBA.4/5 booster. FIG. 40 , among other things, presents calculatedvariant/Wuhan ref GMT ratios, which indicate that:

-   -   (i) BNT162b2 booster results in relatively poor        cross-neutralization, especially of BA.2 and descendants        (BA.2.12.1, BA.4/5)    -   (ii) BA.1 booster results in superior cross-neutralization of        BA.1, but still relatively poor neutralization of BA.2.12.1,        BA.4/5    -   (iii) BA.4/5 booster results in balanced pan-Omicron        neutralization with very encouraging neutralization against        BA.2, BA.2.12.1 and BA.4/5

Bivalent boosters showed a similar but attenuated trend compared to themonovalent VOC boosters; among bivalent boosters the b2+BA.4/5combination was most effective in driving broad cross-neutralization;the trivalent booster (b2+BA.1+BA.4/5) elicited comparablecross-neutralization to the BA.1/BA.4/5 booster.

-   -   Mice can also be administered two RNA molecules, where the ratio        of the two RNA molecules is not 1:1. For example, mice can be        administered a bivalent vaccine comprising BNT162b2 and BA.4/5        at a ratio of 1:2 (e.g., by administering 0.33 ug of BNT162b2        and 0.66 ug of BA.4/5). Mice can also be administered a bivalent        vaccine comprising BNT162b2 and BA.4/5 at a ratio of 1:3 (e.g.,        by administering 0.25 ug of BNT162b2 and 0.75 ug of BA.4/5).        Such compositions can be administered to vaccine naïve mice or        to mice previously vaccinated with BNT162b2 (e.g., previously        administered two doses for 1 ug of BNT162b2).    -   The present specification demonstrates remarkable efficacy of        BA.4/5 immunization (and specifically of BNT162b2+BA.4/5        immunization, e.g., with sequences provided herein).        Furthermore, the present specification demonstrates efficacy of        BA.4/5 immunization in monovalent, bivalent, and trivalent        formats, and documents surprising efficacy of monovalent BA.4/5.    -   The present disclosure specifically demonstrates remarkable        usefulness of one or more BA.4/5 doses administered to subjects        who have previously been immunized (e.g., with a Wuhan vaccine,        such as with at least (or exactly) two doses of a Wuhan vaccine.

Without wishing to be bound by any particular theory, the presentdisclosure teaches that immunological characteristics of the omicronBA.4/5 spike may render it particularly useful or effective forimmunization of subjects, including those who have been immunized (e.g.,via prior administration of one or more vaccine doses and/or by priorinfection) with the Wuhan strain (and/or with one or more strainsimmunologically related to the Wuhan strain), including specifically byvaccination with one or more (e.g., 1, 2, 3, 4 or more) doses oforiginal BNT162b2.

Example 17: Omicron BA.2 Breakthrough Infection EnhancesCross-Neutralization of BA.2.12.1 and BA.4/BA.5

The present Example 17 is an extension of Example 14, and describesexperiments in which serum samples collected from BA.1- andBA.2-breakthrough cases were analyzed for neutralization activityagainst Omicron BA.4 and BA.5 variants. In addition to confirming theresults described in Example 14, the present Example 17 also providesfurther characterization of antibody responses induced by BA.1 and BA.2breakthrough infections, and provides insights as to what aspects maycontribute to increased neutralization of a BA.4/5 Omicron variant asobserved in a BA.2 breakthrough infection, as compared to a BA.1breakthrough infection. In particular, the present Example demonstratesthat a BA.2 breakthrough infection can induce higher titers ofneutralization antibodies that bind the N-terminal domain (NTD) of aSARS-CoV-2 S protein (e.g., a BA.4/5 SARS-CoV-2 Omicron variant), whichcan result in increased neutralization of a BA.4/5 Omicron variant.

As demonstrated in the previous examples, individuals previouslyadministered RNA encoding a SARS-CoV-2 S protein from a Wuhan strain,who subsequently had an Omicron BA.1 breakthrough infection, have strongserum neutralizing activity against Omicron BA.1, BA.2, and previousSARS-CoV-2 variants of concern (VOCs), yet less against highlycontagious Omicron sublineages BA.4 and BA.5 that have displacedprevious variants. As the latter sublineages are derived from OmicronBA.2, the serum neutralizing activity of COVID-19 mRNA vaccinetriple-immunized individuals who experienced BA.2 breakthrough infectionwas analyzed. The present Example demonstrates that sera of theseindividuals have broadly neutralizing activity against previous VOCs aswell as all tested Omicron sublineages, including BA.2 derived variantsBA.2.12.1, BA.4/BA.5 (confirming the results of previous Example 14).Furthermore, applying antibody depletion the present Example shows thatneutralization of BA.2 and BA.4/BA.5 sublineages by BA.2 convalescentsera is driven to a significant extent by antibodies targeting theN-terminal domain (NTD) of the spike glycoprotein, whereas theirneutralization by Omicron BA.1 convalescent sera depends exclusively onantibodies targeting the receptor binding domain (RBD). These findingssuggest that exposure to Omicron BA.2, in contrast to BA.1 spikeglycoprotein, triggers significant NTD specific recall responses invaccinated individuals and thereby enhances the neutralization ofBA.4/BA.5 sublineages. Given the current epidemiology with apredominance of BA.2 derived sublineages like BA.4/BA.5 and rapidlyongoing evolution, these findings are of high relevance for thedevelopment of Omicron adapted vaccines.

Introduction

Emergence of the SARS-CoV-2 Omicron variant of concern (VOC) in November2021 (Ref. 1) can be considered a turning point in the COVID-19pandemic. Omicron BA.1, which is significantly altered in the spike (S)glycoprotein receptor binding domain (RBD) and N-terminal domain (NTD)relative to an S protein from a Wuhan strain, partially escapespreviously established immunity (Ref. 2). The loss of many epitopes(Refs. 3, 4) decreased susceptibility to neutralizing antibodies inducedby wild-type strain (Wuhan-Hu-1) S glycoprotein-based vaccines or byinfection with previous strains (Refs. 5-7), necessitating a thirdvaccine dose to establish full immunity (Refs. 8-10). Omicron BA.1 wasdisplaced by the BA.2 variant, which in turn was displaced by itsdescendants BA.2.12.1, BA.4 and BA.5 that now dominate in many regions(Refs. 11-14).

Antigenically, BA.2.12.1 exhibits high similarity with BA.2 but notBA.1, whereas BA.4 and BA.5 differ considerably from BA.2 and even moreso from BA.1, in line with their genealogy (Refs. 15 and 16). While someamino acid changes in the RBD are shared between all Omicronsub-lineages, the alteration L452Q is only found in BA.2.12.1 and is theonly residue which distinguishes its RBD from that of the BA.2 variant.The L452R and F486V alterations are BA.4/BA.5-specific, whereas S371F,T376A, D405N, and R408S are shared by BA.2 and its descendants BA.2.12.1and BA.4/BA.5, but not BA.1 (FIG. 33 ). These amino acid exchanges areassociated with further escape from vaccine-induced neutralizingantibodies and therapeutic antibody drugs targeting the wild-type Sglycoprotein (Refs. 6, 15, 17-20). The NTDs of BA.2 and its descendantsare antigenically closer to the wild-type strain and lack several aminoacid changes, insertions, and deletions that occurred in BA.1 (FIG. 33). For instance, Δ143-145, L212I, or ins214EPE, which rendered the BA.1variant resistant to a panel of NTD-directed monoclonal antibodiesraised against the wild-type S glycoprotein, are not found in BA.2 anddescendants (Refs. 21, 22).

As demonstrated in the previous Examples, Omicron BA.1 breakthroughinfection of BNT162b2 vaccinated individuals augments broadlyneutralizing activity against Omicron BA.1, BA.2 and previous VOCs atlevels similar to those observed against SARS-CoV-2 wild-type. BA.1breakthrough infection of triple BNT162b2-vaccinated individuals inducesa robust recall response, primarily expanding memory B cells againstepitopes shared broadly amongst variants, rather than inducing B cellsspecific to BA.1 only. Neutralization of Omicron sublineages BA.4 andBA.5 was increased to a lesser extent in BA.1 breakthrough patients ascompared to the BA.1 variant, and geometric mean titers were comparableto those against the phylogenetically more distant SARS-CoV-1.

Given that Omicron BA.2 is more closely related to BA.4/BA.5 than toBA.1, whether BA.2 breakthrough infection would shiftcross-neutralization activity more towards these most recent Omicronsublineages, as compared to BA.1 breakthrough infection, was assessed.The neutralization of different Omicron sublineages by serum samples wascompared from three different cohorts of individuals triple-vaccinatedwith mRNA COVID-19 vaccines, namely from individuals with no history ofSARS-CoV-2 infection and individuals that experienced breakthroughinfection with either BA.1 or BA.2. In addition, the contribution ofserum antibodies targeting the S glycoprotein RBD versus the NTD toOmicron sublineage neutralization was characterized. The resulting dataincrease current understanding on Omicron immune escape mechanisms andthe effects of immunization on variant cross-neutralization, and thushelp guide further vaccine development.

Results

Cohorts and Sampling

This study investigated serum samples from three cohorts: from BNT162b2triple-vaccinated individuals who were SARS-CoV-2-naïve at the time ofsampling (BNT162b2³, n=18), from individuals vaccinated with three dosesof mRNA COVID-19 vaccine (BNT162b2/mRNA-1273 homologous or heterologousregimens) who subsequently had a breakthrough infection with Omicron ata time of BA.1 dominance (mRNA-Vax³+BA.1, n=14), or from triple mRNAvaccinated individuals with a breakthrough infection at a time of BA.2dominance (mRNA-Vax³+BA.2, n=13). For convalescent cohorts, relevantintervals between key events such as the most recent vaccination andinfection are provided in FIG. 41 . Sera were derived from the biosamplecollections of BNT162b2 vaccine trials and from a non-interventionalstudy researching vaccinated patients that had experienced Omicronbreakthrough infection.

Omicron BA.2 Breakthrough Infection of Triple mRNA-VaccinatedIndividuals Induces Broad Neutralization of VOCs Including OmicronBA.4/BA.5

Neutralizing activity of immune sera was tested in a well-characterizedpseudovirus neutralization test (pVNT) (Refs. 24, 25) by determining 50%pseudovirus neutralization (pVN₅₀) geometric mean titers (GMTs) withpseudoviruses bearing the S glycoproteins of the SARS-CoV-2 wild-typestrain, or Alpha, Beta, Delta, Omicron BA.1, BA.2, and the BA.2-derivedsublineages BA.2.12.1, BA.4 and BA.5. As BA.4 and BA.5 share anidentical S glycoprotein sequence, in the present Example they arereferred to as BA.4/5 in the context of the pVNT. In addition, SARS-CoV(herein referred to as SARS-CoV-1) was assayed to detect potentialpan-Sarbecovirus neutralizing activity (Ref. 26). As an orthogonal testsystem, a live SARS-CoV 2 neutralization test (VNT) was also used thatanalyzes neutralization during multicycle replication of authentic virus(SARS-CoV-2 wild-type strain and VOCs including BA.4, except OmicronBA.2.12.1) with the antibodies present during the entire test period.

In the pVNT, sera from all three cohorts robustly neutralized thewild-type strain, Alpha, Beta, Delta VOCs as well as Omicron BA.1 andBA.2 lineages with neutralization activity being more pronounced in thebreakthrough infected individuals, particularly in the BA.1 breakthroughinfection cohort (mRNA-Vax3+BA.1). However, serum neutralizing activityof BNT162b2 triple-vaccinated SARS-CoV-2 naïve (BNT162b2³) andmRNA-Vax³+BA.1 individuals against BA.2.12.1 was significantly reducedcompared to wild-type (p<0.05) and even more so for BA.4/5(p<0.001; >5-fold compared to the wild-type strain) (FIG. 42(A)). Incontrast, sera from triple mRNA-vaccinated individuals with Omicron BA.2breakthrough infection (mRNA-Vax3+BA.2) neutralized the BA.2.12.1pseudovirus as robustly as the wild-type strain. Neutralization ofBA.4/5 was broadly similar to that of BA.2.12.1, and the reductionrelative to the wild-type strain significant (p<0.05) yet lesspronounced (˜2.5-fold) as compared to the two other cohorts.

To compare the cohorts with regard to neutralization breadthirrespective of the magnitude of antibody titers, the VOC pVN₅₀ GMTs wasnormalized against the Wuhan strain. The ratios showed that BA.4/5cross-neutralization was substantially stronger in mRNA-Vax³+BA.2 (GMTratio 0.38) as compared to mRNA-Vax³+BA.1 and BNT162b2³ sera (GMT ratios0.18 and 0.17) (FIG. 42(B)). Similarly, cross-neutralization of OmicronBA.2.12.1 by mRNA-Vax³+BA.2 sera (GMT ratio 0.52) was stronger than bymRNA-Vax³+BA.1 sera (GMT ratio 0.43), and even more so than by BNT162b2³sera (GMT ratio 0.26).

A separate analysis including only the BNT162b2 vaccinated individualswithin those three cohorts confirmed that BA.2 breakthrough infection isassociated with considerable BA.4/5 cross-neutralization (BA.4/5 towild-type GMT ratio 0.42), whereas after BA.1 breakthrough infectionpVN₅₀ GMTs against BA.4/5 were ˜6-fold lower than those againstwild-type (i.e., GMT ratio 0.17) (FIG. 45 ((A)-(C)).Cross-neutralization of BA.2 and BA.2.12.1 by sera of the BA.1 or BA.2convalescents was superior to that of BNT162b2 triple-vaccinatedSARS-CoV-2 naïve individuals.

The authentic live SARS-CoV-2 virus neutralization assay provided VOCneutralizing titers that strongly correlated with those from the pVNTassay (FIG. 46 ) and confirmed the major findings in FIG. 42 . In thisassay, 50% virus neutralization (VN₅₀) GMT against Omicron BA.2 inBNT162b2³ sera was strongly reduced compared to that against wild-type(p<0.0001), whereas sera from both convalescent groups exhibited strongneutralizing activity, with VN₅₀ GMTs comparable to those against thewild-type strain (FIG. 43(A)). Reduction of neutralizing activityagainst Omicron BA.4 was less pronounced in the BA.2 convalescent cohortas compared to BNT162b2³ and mRNA-Vax³+BA.1 cohorts (VN₅₀ GMTs ˜2.5-foldas compared to ˜15-fold and 5-fold lower than against the wild-typestrain, respectively).

In line with the pVNT data, magnitude-independent analyses via thecalculated ratios of VOC VN₅₀ GMTs against the wild-type strain showedthat BA.4 cross-neutralization was stronger in the mRNA-Vax³+BA.2 cohort(GMT ratio 0.39) as compared to the mRNA-Vax³+BA.1 (GMT ratio 0.20) andBNT162b2³ (GMT ratio 0.07) cohorts (FIG. 43(B)) and similarly so withinthe sub-cohort of BNT162b2 triple-vaccinated individuals (FIGS.45(D)-(F)).

In aggregate, these data demonstrate that Omicron BA.2 breakthroughinfection of vaccinated individuals was associated with broadneutralizing activity against all tested Omicron-sublineages andprevious SARS-CoV-2 VOCs. In particular, these data indicate thatbreakthrough infection with BA.2 was more effective (˜2-fold highercross neutralization) than that with BA.1 at refocusing neutralizingantibody responses towards the BA.4/BA.5 S glycoprotein.

Neutralization of Omicron BA.2 and BA.4/5 by sera of triple mRNAvaccinated BA.2 convalescent individuals is mediated to a large extentby NTD-targeting antibodies.

To dissect the role of serum antibodies binding either to the RBD or theNTD of the S glycoprotein for neutralization of SARS-CoV-2 wild-type,Omicron BA.1, BA.2, and BA.4/5, these antibody fractions were depletedseparately from sera of the three cohorts (n=6 each, FIG. 47(A)). TheSARS-CoV-2 wild-type strain S glycoprotein RBD and NTD baits was usedfor depletion, as VOC breakthrough infections have been demonstrated topredominantly elicit recall responses recognizing epitopes conservedacross known VOCs (Refs. 10, 23, and 27). The depletion experimentsremoved >97% of all RBD-binding antibodies and >74% of all NTD-bindingantibodies (FIG. 47(B)). Depleted sera were subsequently tested in pVNTassays. RBD-antibody depletion strongly diminished neutralizing activityagainst the wild-type strain in sera from all cohorts, whereasneutralizing activity was mostly retained (>80% remaining activity) upondepletion of NTD-binding antibodies (FIG. 44 (A)). Neutralization ofOmicron BA.1 was completely abrogated upon depletion of RBD-bindingantibodies and largely unaffected by NTD-binding antibody depletion. Forneutralization of BA.2, RBD-antibody depletion almost completelyabolished neutralizing activity of mRNA-Vax³+BA.1 sera (about 2%residual neutralization activity). The reduction of neutralizing titersfor BNT162b2³ and particularly mRNA-Vax³+BA.2 sera was less severe with˜12% and ˜24% remaining neutralizing activity, respectively. Incontrast, depletion of NTD-binding antibodies did not considerablyimpact the neutralizing activity of BNT162b2³ and mRNA-Vax³+BA.1 sera(˜91 and ˜99% of undepleted control, respectively), while neutralizingactivity of mRNA-Vax³+BA.2 sera was reduced to ˜50%. A similar patternwas seen following RBD-antibody depletion for neutralization of BA.4/5,with strongly reduced neutralizing activity of mRNA-Vax³+BA.1 sera (˜3%residual activity) versus less severe reductions for BNT162b2³ andmRNA-Vax³+BA.2 sera (˜20 and ˜26% remaining activity, respectively).Depletion of NTD-binding antibodies had a larger impact for BA.4/5neutralization compared to BA.2, with remaining neutralizing activity ofBNT162b2³ and mRNA-Vax³+BA.1 sera of ˜70 and ˜90% respectively, againwith the strongest effect (˜48% of undepleted control) of mRNA-Vax³+BA.2sera.

As an orthogonal approach the neutralizing activity of sera was assessedfrom those 3 cohorts of vaccinated individuals against a pseudovirusharboring an engineered hybrid S glycoprotein consisting of the OmicronBA.1 N-terminus including the NTD (amino acids 1-338) and the BA.4/5 Cterminus including the RBD.

The pVN₅₀ GMT against the Omicron BA.1-BA.4/5 hybrid pseudovirus in serafrom BNT162b2³ was moderately below (1.86-fold) the GMT for the BA.4/5pseudovirus, and in the BA.1 convalescents the GMT was only marginallyaffected (<1.5-fold reduction) (FIG. 44(B)). In contrast, in BA.2convalescent sera titers against the hybrid pseudovirus wereconsiderably lower than those against the BA.4/5 pseudovirus (>3-foldreduction of GMT) (FIG. 44(B)), suggesting that substantial neutralizingactivity can be attributed to NTD epitopes that are shared betweenOmicron BA.2 and BA.4/5.

In aggregate the data obtained in both experiments indicate that acrossall these VOCs RBD-binding antibodies provided a major contribution toneutralization. Additionally, exposure to BA.1 (that differssubstantially from previous VOCs in its NTD; FIG. 33 ) boosted recallresponses of vaccine-induced neutralizing antibodies that primarily bindthe RBD, whereas exposure to BA.2 S glycoprotein (with an NTD closerrelated to previous VOCs) can build on existing memory and elicited aconsiderable recall of NTD-targeting antibodies that in turn contributedsubstantially to the neutralization of BA.2 and BA.4/5.

Discussion

Recent studies have demonstrated that Omicron BA.1 breakthroughinfection in individuals vaccinated with mRNA vaccines BNT162b2 ormRNA-1273 or an inactivated virus vaccine boosts serum neutralizingtiters against VOCs including BA.2 (Refs. 10, 15, 23), but not againstBA.2.12.1 or BA.4/BA.5. The immune escape has been attributed toboosting of pre-existing neutralizing antibody responses that recognizeepitopes shared between the SARS-CoV-2 wild-type strain and Omicron BA.1but are in part absent in BA.2.12.1, BA.4, and BA.5 due to alterationsat key residues including L452Q/L452R, and F486V (Ref. 15).

In the present Example, BA.2 breakthrough infection was associated withbroadly neutralizing activity including BA.2 and its descendantsBA.2.12.1, BA.4 and BA.5. These findings suggest that the highersequence similarity of BA.2 with BA.2.12.1 and BA.4/5 in the Sglycoprotein RBD as well as the NTD drives more efficientcross-neutralization as compared to breakthrough infections with theantigenically more distant BA.1 variant. In particular, BA.1breakthrough infection may not elicit a strong recall of NTD-specificmemory B cells owing to the substantial alterations within the BA.1 NTD(FIG. 33 ) given that breakthrough infection with heterologousSARS-CoV-2 strains primarily expands a memory B cell repertoire againstconserved S glycoprotein epitopes (Refs. 10, 23). The data describedherein that was obtained in antibody-depletion and hybrid pseudovirusexperiments show that NTD-binding antibodies have a substantialcontribution to neutralizing activity against Omicron BA.4/5 intriple-vaccinated BA.2 convalescent sera, whereas neutralizing activityin BA.1 convalescent sera largely relies on RBD-binding antibodies. Thisfinding is consistent with the observation that NTD-binding antibodiesisolated from BA.2 breakthrough infected individuals do not neutralizeBA.1 (Ref. 29). Together these important findings extend our knowledgeon how vaccinations and boosters with the current wild-type strain-basedvaccines together with breakthrough infections with the various VOCsshape the immunity patterns within the population and are material toinform further vaccine development and adaptation in response to currentand emerging VOCs.

Notwithstanding the importance of vaccination with currently approvedwild-type-strain based vaccines such as BNT162b2 that offer effectiveprotection from severe disease by current VOCs including Omicron BA.1and BA.2 (Refs. 30 and 31), the present findings highlight thatconsideration of rapidly evolving epidemiological landscapes and newlyemerging SARS-CoV-2 variants is important for guiding vaccine adaptationprograms. For instance, while the efficacy of vaccine adaptation to theBA.1 strain S glycoprotein sequence is currently under investigation inclinical trials, the present data suggest that further benefit may bederived from a vaccine adapted to the sequence of BA.2 or descendants.

Materials and Methods

Study Design, Recruitment of Participants and Sample Collection

The objective of this study was to investigate the effect of OmicronBA.2 breakthrough infection on the cross-variant neutralization capacityof human sera. Immune responses in triple-mRNA(BNT162b2/mRNA-1273)-vaccinated individuals with a confirmed subsequentSARS-CoV-2 breakthrough infection in a period of Omicron BA.2lineage-dominance in Germany (March to May 2022; mRNA-Vax3+BA.2), wascompared to that of triple-mRNA-vaccinated individuals with a confirmedsubsequent SARS-CoV-2 breakthrough infection in a period of Omicron BA.1lineage-dominance (November 2021 to mid-January 2022; mRNA-Vax³+BA.1)(Refs. 1 and 2) and triple-BNT162b2-vaccinated individuals that wereSARS-CoV-2-naïve (nucleocapsid seronegative) at the time of samplecollection (BNT162b2³). Serum neutralizing capability was characterizedusing pseudovirus and live SARS-CoV-2 neutralization assays. Data forthe reference cohorts BNT162b2³ and mRNA-Vax³+BA.1 were previouslypublished (Ref. 10), except for newly generated BA.2.12.1 neutralizationdata. Cross-neutralization of variants was further characterized insmaller sub-cohorts after depletion of either wild-type S glycoproteinNTD- or RBD-targeted neutralizing antibodies. Individuals from theBNT162b2³ cohort provided informed consent as part of theirparticipation in the Phase 2 trial BNT162-17 (NCT05004181). Participantsfrom the mRNA-Vax³+Omi BA.1 and mRNA-Vax³+BA.2 cohorts were recruitedfrom University Hospital, Goethe University Frankfurt as part of anon-interventional study (protocol approved by the Ethics Board of theUniversity Hospital [No. 2021-560]) researching patients that hadexperienced Omicron breakthrough infection following vaccination forCOVID-19. Omicron BA.1 infections were confirmed with variant-specificPCR. The infections of 4 BA.1 convalescent participants in this studywere further characterized by genome sequencing. In all 4 cases, genomesequencing confirmed Omicron BA.1 infection.

Demographic and clinical data for all participants and samplingtimepoints are provided (FIG. 41 ). All participants had no documentedhistory of SARS-CoV-2 infection prior to vaccination. Participants werefree of symptoms at the time of blood collection.

Serum was isolated by centrifugation of drawn blood at 2000×g for 10minutes and cryopreserved until use.

VSV-SARS-CoV-2 S Variant Pseudovirus Generation

A recombinant replication-deficient vesicular stomatitis virus (VSV)vector that encodes green fluorescent protein (GFP) and luciferaseinstead of the VSV-glycoprotein (VSV-G) was pseudotyped with SARS-CoV-1S glycoprotein (UniProt Ref: P59594) and with SARS-CoV-2 S glycoproteinderived from either the Wuhan-Hu-1 reference strain (NCBI Ref:43740568), the Alpha variant (alterations: Δ69/70, Δ144, N501Y, A570D,D614G, P681H, T716I, S982A, D1118H), the Beta variant (alterations:L18F, D80A, D215G, Δ242-244, R246I, K417N, E484K, N501Y, D614G, A701V),the Delta variant (alterations: T19R, G142D, E156G, Δ157/158, K417N,L452R, T478K, D614G, P681R, D950N), the Omicron BA.1 variant(alterations: A67V, Δ69/70, T95I, G142D, Δ143-145, Δ211, L212I,ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N,T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y,N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F), the OmicronBA.2 variant (alterations: T19I, Δ24-26, A27S, G142D, V213G, G339D,S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, S477N, T478K,E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K,D796Y, Q954H, N969K), the Omicron BA.2.12.1 variant (alterations: T19I,Δ24-26, A27S, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N,R408S, K417N, N440K, L452Q, S477N, T478K, E484A, Q493R, Q498R, N501Y,Y505H, D614G, H655Y, N679K, P681H, S704L, N764K, D796Y, Q954H, N969K),the Omicron BA.4/5 variant (alterations: T19I, Δ24-26, A27S, Δ69/70,G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N,N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G,H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K), or an artificialOmicron BA.1-BA.4/5 hybrid S glycoprotein (alterations: A67V, Δ69/70,T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D, S371F, S373P,S375F, T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A,F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y,Q954H, N969K) according to published pseudotyping protocols (Ref. 3). Adiagram of SARS-CoV-2 S glycoprotein alterations is shown in FIG. 48 anda separate alignment of S glycoprotein alterations in Omicronsub-lineages is displayed in FIG. 33 . In brief, HEK293T/17 monolayers(ATCC® CRL-11268™) cultured in Dulbecco's modified Eagle's medium (DMEM)with GlutaMAX™ (Gibco) supplemented with 10% heat-inactivated fetalbovine serum (FBS [Sigma-Aldrich]) (referred to as medium) weretransfected with Sanger sequencing-verified SARS-CoV-1 orvariant-specific SARS-CoV-2 S expression plasmid with Lipofectamine LTX(Life Technologies) following the manufacturer's instructions. At 24hours after transfection, the cells were infected at a multiplicity ofinfection (MOI) of three with VSV-G complemented VSVΔG vector. Afterincubation for 2 hours at 37° C. with 7.5% CO₂, cells were washed twicewith phosphate buffered saline (PBS) before medium supplemented withanti-VSV-G antibody (clone 8G5F11, Kerafast Inc.) was added toneutralize residual VSV-G-complemented input virus. VSV-SARS-CoV-2-Spseudotype-containing medium was harvested 20 hours after inoculation,passed through a 0.2 μm filter (Nalgene) and stored at −80° C. Thepseudovirus batches were titrated on Vero 76 cells (ATCC® CRL-1587™)cultured in medium. The relative luciferase units induced by a definedvolume of a SARS-CoV-2 wild-type strain S glycoprotein pseudovirusreference batch previously described in Muik et al., 2021 (Ref. 4), thatcorresponds to an infectious titer of 200 transducing units (TU) per mL,was used as a comparator. Input volumes for the SARS-CoV-2 variantpseudovirus batches were calculated to normalize the infectious titerbased on the relative luciferase units relative to the reference.

Pseudovirus Neutralization Assay

Vero 76 cells were seeded in 96-well white, flat-bottom plates (ThermoScientific) at 40,000 cells/well in medium 4 hours prior to the assayand cultured at 37° C. with 7.5% CO₂. Each individual serum was seriallydiluted 2-fold in medium with the first dilution being 1:5(SARS-CoV-2-naïve triple BNT162b2 vaccinated; dilution range of 1:5 to1:5,120) or 1:30 (triple vaccinated after subsequent Omicron BA.1 orBA.2 breakthrough infection; dilution range of 1:30 to 1:30,720). In thecase of the SARS-CoV-1 pseudovirus assay, the serum of all individualswas initially diluted 1:5 (dilution range of 1:5 to 1:5,120).VSV-SARS-CoV-2-S/VSV-SARS-CoV-1-S particles were diluted in medium toobtain 200 TU in the assay. Serum dilutions were mixed 1:1 withpseudovirus (n=2 technical replicates per serum per pseudovirus) for 30minutes at room temperature before being added to Vero 76 cellmonolayers and incubated at 37° C. with 7.5% CO₂ for 24 hours.Supernatants were removed and the cells were lysed with luciferasereagent (Promega). Luminescence was recorded on a CLARIOstar® Plusmicroplate reader (BMG Labtech), and neutralization titers werecalculated as the reciprocal of the highest serum dilution that stillresulted in 50% reduction in luminescence. For depletion studiesresolution with regards to neutralization titers was increased, in orderto discriminate smaller than 2-fold differences on an individual serumlevel. Neutralization titers were determined by generating a 4-parameterlogistical (4PL) fit of the percent neutralization at each serial serumdilution. The 50% pseudovirus neutralization (pVN₅₀) titer was reportedas the interpolated reciprocal of the dilution yielding a 50% reductionin luminescence. Results for all pseudovirus neutralization experimentswere expressed as geometric mean titers (GMT) of duplicates. If noneutralization was observed, an arbitrary titer value of half of thelimit of detection [LOD] was reported. SARS-CoV-2 wild-type strain, andAlpha, Beta, Delta, BA.1, BA.4/5 VOC, as well as SARS-CoV-1 pseudovirusneutralizing GMTs for the SARS-CoV-2 naïve BNT162b2 triple-vaccinatedcohort and the triple-vaccinated BA.1 convalescent cohort werepreviously reported in Quandt. et al. (Ref. 10). Only the BA.2.12.1neutralization data was newly generated from serum samples for thisstudy.

Live SARS-CoV-2 Neutralization Assay

SARS-CoV-2 virus neutralization titers were determined by amicroneutralization assay based on cytopathic effect (CPE) at VisMederiS.r.l., Siena, Italy. In brief, heat-inactivated serum samples fromindividuals were serially diluted 1:2 (starting at 1:10; n=2 technicalreplicates per serum per virus) and incubated for 1 hour at 37° C. with100 TCID50 of the live wild-type-like SARS-CoV-2 virus strain2019-nCOV/ITALY-INMI1 (GenBank: MT066156), Alpha virus strain nCoV19isolate/England/MIG457/2020 (alterations: Δ69/70, Δ144, N501Y, A570D,D614G, P681H, T716I, S982A, D1118H), Beta virus strain nCoV19isolate/England ex-SA/HCM002/2021 (alterations: D80A, D215G, Δ242-244,K417N, E484K, N501Y, D614G, A701V), sequence-verified Delta strainisolated from a nasopharyngeal swab (alterations: T19R, G142D, E156G,Δ157/158, L452R, T478K, D614G, P681R, R6820, D950N), Omicron BA.1 strainhCoV-19/Belgium/rega-20174/2021 (alterations: A67V, Δ69/70, T95I, G142D,Δ143-145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N,N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H,T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K,L981F), sequence-verified Omicron BA.2 strain (alterations: T19I,Δ24-26, A27S, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S,K417N, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y,N679K, P681H, R682W, N764K, D796Y, Q954H, N969K), or sequence-verifiedOmicron BA.4 strain (alterations: V3G, T19I, Δ24-26, A27S, Δ69/70,G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N,N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G,H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K) to allow anyantigen-specific antibodies to bind to the virus. A diagram of Sglycoprotein alterations is shown in FIG. 48 . The 2019-nCOV/ITALY-INMI1strain S glycoprotein is identical in sequence to the wild-typeSARS-CoV-2 S (Wuhan-Hu-1 isolate). Vero E6 (ATCC® CRL-1586™) cellmonolayers were inoculated with the serum/virus mix in 96-well platesand incubated for 3 days (2019-nCOV/ITALY-INMI1 strain) or 4 days(Alpha, Beta, Delta, Omicron BA.1, BA.2 and BA.4 variant strain) toallow infection by non-neutralized virus. The plates were observed underan inverted light microscope and the wells were scored as positive forSARS-CoV-2 infection (i.e., showing CPE) or negative for SARS-CoV-2infection (i.e., cells were alive without CPE). The neutralization titerwas determined as the reciprocal of the highest serum dilution thatprotected more than 50% of cells from CPE and reported as GMT ofduplicates. If no neutralization was observed, an arbitrary titer valueof 5 (half of the LOD) was reported.

Depletion of RBD- or NTD-Binding Antibodies from Human Sera

SARS-CoV-2 wild-type strain S glycoprotein RBD- and NTD-coupled magneticbeads (Acro Biosystems, Cat. no. MBS-K002 and MBS-K019; 40 μg RBD/mgbeads and 38 μg NTD/mg beads, respectively) were prepared according tothe manufacturer's instructions. Beads were resuspended in ultrapurewater at 1 mg beads/mL and a magnet was used to collect and wash thebeads with PBS. Beads were resuspended in serum to obtain 20 μg RBD- orNTD-bait per 100 μL serum. A mock depletion (undepleted control) wasperformed for each serum by adding 0.5 mg Biotin-saturated MyOne™Streptavidin T1 Dynabeads™ (ThermoFisher, Cat. no. 65601) per 100 μLserum. Beads were incubated with human sera for 1 hour with gentlerotation. A magnet was used to separate bead-bound antibodies from thedepleted supernatant. Depleted and undepleted sera were analyzed forcross-neutralization capacity using pseudovirus neutralization assays.Depletion efficacy for both RBD- and NTD-binding antibodies wasdetermined by a multiplexed electrochemiluminescence immunoassay (MesoScale Discovery, V-Plex SARS-CoV-2 Panel 1 Kit, Cat. No. K15359U-2).

Statistical Analysis

The statistical method of aggregation used for the analysis of antibodytiters is the geometric mean and for the ratio of SARS-CoV-2 VOC titerand wild-type strain titer the geometric mean and the corresponding 95%confidence interval. The use of the geometric mean accounts for thenon-normal distribution of antibody titers, which span several orders ofmagnitude. The Friedman test with Dunn's correction for multiplecomparisons was used to conduct pairwise signed-rank tests of groupgeometric mean neutralizing antibody titers with a common control group.Spearman correlation was used to evaluate the monotonic relationshipbetween non-normally distributed datasets. All statistical analyses wereperformed using GraphPad Prism software version 9.

REFERENCES FOR EXAMPLE 17

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Example 18: Exposure to BA.4/BA.5 Spike Glycoprotein Drives Pan-OmicronNeutralization in Vaccine-Experienced Humans and Mice

The present Example is an extension of Examples 7, 13, 16, and 17, anddescribes (i) immune response data from human subjects previouslyvaccinated against SARS-CoV-2 who have had a BA.4/5-breakthroughinfection (in the present example, human subjects who have previouslybeen administered three doses of RNA vaccine(s) encoding the SARS-CoV-2S protein of a Wuhan variant), and (ii) further mouse data, showingimmune responses induced by RNA encoding SARS-CoV-2 S proteinscomprising mutations characteristic of certain Omicron variants.

Abstract

The SARS-CoV-2 Omicron variant and its sublineages show pronounced viralescape from neutralizing antibodies owing to numerous amino acidalterations within the spike (S) glycoprotein. As demonstrated in theprevious Examples, breakthrough infection of vaccinated individuals withOmicron sublineages BA.1 and BA.2 has been associated with distinctpatterns of cross-neutralizing activity against SARS-CoV-2 variants ofconcern (VOCs), most notably against the recently emerged BA.2descendants Omicron BA.4 and BA.5. Here, the effect of Omicron BA.4/BA.5S glycoprotein exposure on the magnitude and breadth of the neutralizingantibody response is investigated in vaccine-experienced humans andmice. The immune sera from triple mRNA-vaccinated individuals withOmicron BA.4/BA.5 breakthrough infection is shown to display broad androbust neutralizing activity against Omicron BA.1, BA.2, BA.2.12.1, andBA.4/BA.5. Administration of a BA.4/BA.5-adapted mRNA booster vaccine tomice following SARS-CoV-2 wild-type (Wuhan) strain-based primaryimmunization is associated with similarly broad neutralizing activity.Immunization of vaccine-naïve mice with a bivalent mRNA vaccine encodingBA.4/BA.5-adapted and wild-type-based S glycoprotein immunogens isfurther shown to induce strong and broad neutralizing activity againstOmicron and non-Omicron VOCs. These findings support the conclusionthat, when administered as monovalent or bivalent boosters, OmicronBA.4/BA.5-adapted vaccines (e.g., RNA encoding a SARS-CoV-2 S proteincomprising mutations characteristic of these variants) can lead toimproved neutralization breadth against current and newly emerging VOCs(improved, in particular, as compared to a BA.1-adapted vaccine). Thesefindings also show that a BA.4/BA.5 vaccine, when administered in abivalent format has the potential to confer protection to subjects withno pre-existing immunity against SARS-CoV-2 (e.g., to exposure-naïvesubjects).

Introduction

The SARS-CoV-2 Omicron variant of concern (VOC) has had a major impacton the epidemiological landscape of the COVID-19 pandemic since itsemergence in November 2021 (Refs. 1-2). Significant alterations in thespike (S) glycoprotein of the initial Omicron variant BA.1 led to theloss of many neutralizing antibody epitopes (Ref. 3) and rendered BA.1capable of partially escaping previously established SARS-CoV-2wild-type strain (Wuhan-Hu-1)-based immunity (Refs. 4-6). Hence,breakthrough infections of vaccinated individuals with Omicron are morecommon than with previous VOCs. While Omicron BA.1 was displaced by theBA.2 variant in many countries around the globe, other variants such asBA.1.1 and BA.3 temporarily and/or locally gained momentum but did notbecome globally dominant (Refs. 7-9). Omicron BA.2.12.1 displaced BA.2to become dominant in the United States in the interim, whereas BA.4 andBA.5 displaced BA.2 in Europe, parts of Africa, and Asia/Pacific (Refs.8, 10-12. Currently the VOCs Omicron BA.4 and BA.5 are predominant inlarge parts of the world, including the United States where theyeventually displaced BA.2.12.1 (Ref. 13).

Omicron lineage VOCs have acquired numerous alterations (amino acidexchanges, insertions, or deletions) in the S glycoprotein, among whichsome are shared between all Omicron VOCs while others are specific toone or more sublineages (see FIG. 33 ). Antigenically, BA.2.12.1exhibits high similarity with BA.2 but not BA.1, whereas BA.4 and BA.5differ considerably from BA.2 and even more so from BA.1, in line withtheir genealogy (Ref. 14). Major differences of BA.1 from the remainingOmicron VOCs include Δ143-145, L212I, or ins214EPE in the S glycoproteinN-terminal domain and G446S or G496S in the receptor binding domain(RBD). Amino acid changes T376A, D405N, and R408S in the RBD are in turncommon to BA.2 and its descendants but not found in BA.1. In addition,some alterations are specific for certain BA.2-descendant VOCs,including L452Q for BA.2.12.1 or L452R and F486V for BA.4 and BA.5. Mostof these shared and sublineage-specific alterations were shown to playan important role in immune escape from monoclonal antibodies andpolyclonal sera raised against the wild-type S glycoprotein (Ref. 15).

As described in the previous Examples, breakthrough infection withOmicron BA.1 or BA.2 of individuals immunized with mRNA vaccines or aninactivated virus vaccine has been associated with potent neutralizingactivity against Omicron BA.1, BA.2 and previous VOCs (see also, Refs.16-21). A considerable boost of Omicron BA.2.12.1 and BA.4/BA.5neutralization relative to SARS-CoV-2 naïve vaccine sera was onlyevident in BA.2 breakthrough cases, with titers against BA.4/BA.5 beinglower than those against the remaining VOCs (see also Refs. 19-21).Administration of an Omicron BA.1-adapted booster to mice afterwild-type strain-based primary immunization has previously been shown toenhance neutralizing activity against BA.1 compared to a wild-typebooster (see previous Example 16 and Ref. 22). Preliminary analyses of aPhase 2 clinical trial have also shown that sera from SARS-CoV-2 naïveindividuals vaccinated with an Omicron BA.1-adapted mRNA vaccine as a4^(th) dose showed significantly increased neutralizing responsesagainst BA.1 compared to sera from individuals boosted with theprototypic mRNA vaccine (Ref. 23). However, the BA.1-adapted booster wasnot associated with increased BA.4/BA.5 cross-neutralization, asresponses against BA.4/BA.5 were approximately three-fold and five-foldlower than responses against BA.1 and the prototypic strain,respectively, in sera from both BA.1 and prototype-boosted individuals.

Given the current predominance of the highly contagious VOCs OmicronBA.4 and BA.5 in large parts of the globe, the present Example wasdesigned to investigate whether exposure to an Omicron BA.4/BA.5 Sglycoprotein would trigger a broader neutralizing antibody responseagainst relevant Omicron sublineages. The breadth of neutralizingactivity was tested against Omicron VOCs in immune sera from vaccinatedindividuals with Omicron BA.4/BA.5 breakthrough infection and in serafrom mice that received Omicron BA.4/BA.5-adapted booster vaccinesfollowing primary immunization with BNT162b2. In addition, the breadthof neutralizing activity was evaluated in sera from mice immunized withOmicron BA.4/BA.5-adapted vaccines without prior exposure to thewild-type S glycoprotein. The data described in the present Exampleprovides further insight into Omicron immune escape mechanisms and theeffects of immunization on variant cross-neutralization, and thus isuseful for guiding selection of vaccination strategies.

Results

Study Design, Cohorts, and Sampling

The effect of Omicron BA.4/BA.5 breakthrough infection in individualsvaccinated with three doses of mRNA COVID-19 vaccine (BNT162b2/mRNA-1273homologous or heterologous regimens) (FIG. 49(A)) and of OmicronBA.4/BA.5-adapted booster vaccination of BNT162b2 pre-immunized mice onthe breadth of neutralizing activity in immune sera (FIG. 49(B)) wasinvestigated. In addition, the effect of primary immunization withOmicron BA.4/BA.5-adapted vaccines was investigated in naïve mice (i.e.,mice without previous exposure to a SARS-CoV-2 S protein, see FIG.49(C)).

For the breakthrough infection study, serum samples were collected fromtriple mRNA vaccinated individuals who subsequently experienced abreakthrough infection with Omicron BA.4 or BA.5 (mRNA-Vax³+BA.4/BA.5,n=17, FIG. 53 ). Three cohorts were included for reference: triple mRNAvaccinated individuals with a breakthrough infection with Omicron BA.2(mRNA-Vax³+BA.2, n=19) or with BA.1 (mRNA-Vax³+BA.2, n=14), and BNT162b2triple-vaccinated individuals who were SARS-CoV-2-naïve at the time ofsampling (BNT162b2³, n=18, FIG. 53 ). Sera were derived the studypreviously described in Examples 7, 13, and 17.

Omicron BA.4/BA.5 Breakthrough Infection of Triple mRNA-VaccinatedIndividuals Results in Pan-Omicron Neutralizing Activity

Neutralizing activity of immune sera was tested in a well-characterizedpseudovirus neutralization test (pVNT) (Refs. 24-25) by determining 50%pseudovirus neutralization (pVN₅₀) geometric mean titers (GMTs) withpseudoviruses bearing the S glycoproteins of the SARS-CoV-2 wild-typestrain or Omicron BA.1, BA.2, and the BA.2-derived sublineagesBA.2.12.1, BA.4 and BA.5. As BA.4 and BA.5 are identical in their Sglycoprotein sequence, in the context of pVNT, BA.4/5 is used. Inaddition, SARS-CoV was assayed (herein referred to as SARS-CoV-1) todetect potential pan-Sarbecovirus neutralizing activity (Ref. 26). As anorthogonal test system, a live SARS-CoV-2 neutralization test (VNT) wasalso used that analyzes neutralization during multicycle replication ofauthentic virus (SARS-CoV-2 wild-type strain and Omicron VOCs BA.1,BA.2, and BA.4) with immune serum present during the entire test period.

In the pVNT assay, sera from the Omicron BA.4/BA.5 breakthroughinfection cohort (mRNA-Vax³+BA.4/BA.5) robustly neutralized thewild-type strain and all tested Omicron VOCs (FIG. 50(A)). The pVN₅₀GMTs against Omicron BA.2 and BA.2.12.1 pseudoviruses were within a2-fold range of the GMT against the wild-type strain (GMTs 613 againstOmicron vs. GMT 1085 against wild-type). Neutralization of BA.1 andBA.4/5 (GMTs 500-521) was broadly similar to that of BA.2, and thereduction relative to the wild-type strain significant (p<0.05) yet alsowithin a ˜2-fold range. The GMT against SARS-CoV-1 was significantlylower (p<0.0001; >50-fold lower than wild-type).

To compare mRNA-Vax³+BA.4/BA.5 to the reference cohorts with OmicronBA.1 or BA.2 breakthrough infection (mRNA-Vax³+BA.1 and mRNA-Vax³+BA.2)and SARS-CoV-2 naïve triple BNT162b2-vaccinated individuals (BNT162b2³),the VOC pVN₅₀ GMTs was normalized against the wild-type strain to allowfor assessment of neutralization breadth irrespective of the magnitudeof antibody titers, which expectedly differs between triple-vaccinatedindividuals with a breakthrough infection and triple-vaccinatedindividuals without infection (Refs. 16 and 21). While BNT162b2³ seramediated considerable cross-neutralization of Omicron BA.1 and BA.2,breakthrough infection with Omicron BA.1 and BA.2 was associated withhigher cross-neutralization of the respective homologous strains (FIG.50(B)). Cross-neutralization of BA.2.12.1, and especially BA.4/5, wasless effective in the mRNA-Vax³+BA.1 cohort (GMT ratios 0.43 and 0.18,respectively) compared to mRNA-Vax³+BA.4/BA.5 (GMT ratios 0.57 and0.48). Cross-neutralization of BA.2.12.1 and BA.4/5 was less reduced inthe mRNA-Vax³+BA.2 cohort (GMT ratios 0.53 and 0.37, respectively).Surprisingly, in the reciprocal case, cross-neutralization of OmicronBA.1 and BA.2 was maintained at comparatively higher levels in themRNA-Vax³+BA.4/BA.5 cohort (GMT ratios 0.46 and 0.57, respectively).Hence, BA.4/BA.5 breakthrough infection resulted in the most efficientcross-neutralization across all tested VOCs (GMT ratios 20.46) of allcohorts evaluated.

The authentic live SARS-CoV-2 virus neutralization assay largelyconfirmed the major pVNT assay findings shown in FIG. 50(A)-(B). 50%virus neutralization (VN₅₀) GMTs in Omicron BA.4/BA.5 breakthrough seraagainst BA.2 and BA.4 were comparable (i.e., within a 1.5-fold range) tothat against the wild-type strain (FIG. 50(C)). Reduction of BA.1neutralization was significant (p<0.01) yet within a 2.5-fold range. TheGMTs normalized against the wild-type strain showed robustcross-neutralization of Omicron BA.1, BA.2, and BA.4 bymRNA-Vax³+BA.4/BA.5 sera (GMT ratio 20.40), whereas BA.4cross-neutralization was considerably less efficient in mRNA-Vax³+BA.1(GMT ratio 0.20) and mRNA-Vax³+BA.2 (GMT ratio 0.39) sera (FIG. 50(D))(Refs. 16, 21). Hence, the findings in both the pVNT and the VNT assaysystem showed that Omicron BA.4/BA.5 breakthrough infection wasassociated with broad neutralizing activity against all Omicronsublineages tested.

Booster Immunization with on Omicron BA.4/BA.5 S Glycoprotein AdaptedmRNA Vaccine Drives Pan-Omicron Neutralization in BNT162b2Double-Vaccinated Mice

The heightened neutralization breadth seen after Omicron BA.4/BA.5breakthrough infection suggested that variant-adapted vaccines based onthe Omicron BA.4/5 S glycoprotein sequence can elicit a recall responsewith broader cross-neutralization than those based on Omicron BA.1. Totest this hypothesis, booster studies were performed inBNT162b2-preimmunized mice (FIG. 49(B)). Mice were administered aprimary series of two immunizations with BNT162b2 on days 0 and 21 and athird dose of either BNT162b2 (1 μg), or a BNT162b2-derivedvariant-adapted vaccine encoding Omicron BA.1 or BA.4/BA.5 Sglycoprotein (FIG. 54 ) on day 104. The adapted vaccines were eitheradministered as monovalent vaccines encoding Omicron BA.1 or BA.4/5 Sglycoprotein (1 μg), or bivalent vaccines comprising BNT162b2 and theOmicron BA.1 or BA.4/5 S glycoprotein adapted vaccine (0.5 μg each).Comparable RNA purity and integrity, and expression of antigens in vitrowere confirmed for BNT162b2 and Omicron-adapted vaccines (FIG.55(A)-(B)).

Neutralizing titers against pseudoviruses expressing the wild-typestrain, Omicron BA.1, BA.2, BA.2.12.1, or BA.4/5 S glycoprotein weredetermined in pVNT assays using sera drawn before the booster (day 104,pre-D3) and on days 7, 21, and 35 after the booster (d7D3, d21D3 andd35D3). The live SARS-CoV-2 neutralization test was used as anorthogonal test system to confirm the observed pseudovirus neutralizingactivity post-boost on d21D3 and d35D3.

Baseline immunization of the mice was assessed by determination ofSARS-CoV-2 pseudovirus neutralizing activity in sera drawn on pre-D3.pVN₅₀ GMTs of the groups dedicated for the various boosters werecomparable, i.e., within a range of 3-fold difference (FIG. 56 ). pVN₅₀GMTs against Omicron BA.1 and BA.2 were 3 to 11-fold lower than thoseagainst the wild-type strain (GMT ratios ≤0.32, FIG. 56(B)). GMTsagainst BA.2.12.1 and BA.4/5 were 10 to 25-fold lower than those againstwild-type (GMT ratios≤0.10).

On d7D3, neutralizing GMTs had increased substantially across groups andagainst all tested variants (FIG. 57 ) with peak titers reached on d21D3(FIG. 51 ). In sera from BNT162b2 boosted mice, strong neutralizingactivity against the wild-type strain was observed, whereas pVN₅₀ GMTsagainst Omicron variants were substantially lower (FIG. 51(A)). TheOmicron BA.1 booster led to comparable neutralization of BA.1 and thewild-type strain, while the pVN₅₀ GMTs against the remaining VOCs wereconsiderably lower. In particular, GMTs against BA.4/5 were reduced13-fold compared to those against wild-type. In contrast, administrationof the BA.4/5 booster resulted in broad neutralizing activity againstall Omicron variants, with pVN₅₀ GMTs comparable (within a 1.5-foldrange) to that against the wild-type strain. Sera from mice thatreceived the BNT162b2/BA.1 bivalent booster had a high pVN₅₀ GMT againstthe wild-type strain, and robust neutralization of Omicron BA.1, whereasthe GMTs against BA.2 and its descendants were slightly lower. TheBNT162b2/Omicron BA.4/5 bivalent booster gave rise to high titersagainst the wild-type strain, comparable to the BNT162b2 monovalentbooster. Omicron neutralization was broadly comparable across allsublineages (within a 2-fold range), with pVN₅₀ GMTs modestly below thatagainst the wild-type strain.

To quantify the booster effect of the third dose of vaccine variants onneutralization of individual VOCs, the fold-changes in pVN₅₀ GMTsdetected on d21D3 was evaluated relative to the baseline GMTs determinedbefore administration of the third dose. BNT162b2 comparably increasedneutralization of all tested variants (pVN₅₀ GMTs 6 to 10-fold higherthan at baseline), whereas the most pronounced effect of the BA.1booster was detected for the neutralization of the homologous VOC(26-fold increase) (FIG. 51(B)). The BA.4/5 booster strongly potentiatedneutralizing activity against BA.2.12.1 and BA.4/5 (>120-fold) and alsohad a substantial effect on BA.1 and BA.2 neutralization (increases of34 and 37-fold, respectively). The BNT162b2/BA.1 and BNT162b2/BA.4/5bivalent vaccines showed patterns of potentiation similar to the BA.1and BA.4/5 monovalent vaccines, respectively, albeit with less focusedincreases in neutralization against the homologous VOCs.

To compare the groups with regard to neutralization breadth irrespectiveof the magnitude, the VOC pVN₅₀ GMTs was normalized against thewild-type strain. The GMT ratios showed that the Omicron BA.4/5 boostervaccine mediated pan-Omicron neutralization (20.65 for all testedvariants) (FIG. 51(C)). In contrast, the BA.1 booster vaccine wasbeneficial for neutralization of BA.1 (GMT ratio 0.77), whereas ratiosfor BA.2 (0.39) and especially for BA.2.12.1 and BA.4/5 (50.16) weresubstantially lower. The bivalent BNT162b2/BA.4/5 vaccine also mediatedbroad neutralizing activity with enhanced cross-neutralization of BA.2,BA.2.12.1 and BA.4/5 compared to the BNT162b2/BA.1 bivalent vaccine,albeit with lower GMT ratios (between 0.27 and 0.46) than the BA.4/5monovalent vaccine.

Again, the authentic live SARS-CoV-2 virus neutralization assay largelyconfirmed the major pVNT assay findings shown in FIG. 51(A)-(C). Serafrom mice administered the BA.4/5 booster dose robustly neutralized allthe variants tested (FIG. 51(D)), thereby confirming the capacity ofthis approach to mediate pan-Omicron neutralization (GMT ratios≥0.39)(FIG. 51(E)). While neutralization of Omicron variants in sera from miceboosted with the BNT162b2/BA.4/5 bivalent vaccine was lower compared tothe BA.4/5 monovalent vaccine, both BA.4/5-containing vaccines exhibitedstronger cross-neutralization of the Omicron variants than theBNT162b2/BA.1 vaccine, and even more so than the monovalent BA.1 vaccineand BNT162b2.

While absolute titers were slightly lower on d7D3 compared to d21D3, theOmicron BA.4/5 vaccine mediated a similarly broad neutralizing activityagainst all variants (FIG. 57 ). BNT162b2/BA.4/5 bivalent boostermediated comparable neutralization breadth (GMT ratios ≥0.35), whereassera from all other booster groups exhibited considerably lowercross-neutralization of BA.2, BA.2.12.1 (and BA.4/5). Similarly, lateranalysis of sera on d35D3 showed substantial potentiation ofneutralizing activity against Omicron variants by the BA.4/5 vaccinebooster compared to baseline, resulting in pan-Omicron neutralization(FIG. 58(A)-(C)). The bivalent BNT162b2/BA.4/5 booster also exhibitedbroad neutralization across all variants tested, whereas the BA.1 andthe BNT162b2/BA.1 vaccines manifested with lower capacity ofcross-neutralization, especially against BA.2.12.1 and BA.4/5.Similarly, in the VNT assay, sera from both groups boosted withBA.4/5-containing vaccines exhibited substantially strongercross-neutralization of all tested Omicron variants compared to theBA.1-containing vaccines and BNT162b2 (FIG. 58(D)-(E)).

These mouse booster results were consistent with those observed inhumans with BA.4/5 breakthrough infections, described above, and furthersuggest that a booster with an Omicron BA.4/5 S glycoprotein adaptedvaccine following primary immunization with a wild-type strain-basedvaccine can elicit pan-Omicron neutralizing activity superior in breadthto a BA.1 S glycoprotein-based booster.

Immunization with an Omicron BA.4/BA.5 S Glycoprotein Adapted mRNAVaccine Drives Pan-Omicron Neutralization in Previously UnvaccinatedMice

Next, experiments were performed to better understand the neutralizingcapacity of serum samples after immunization with the Omicron-adaptedvaccine in mice with no pre-existing immune response against SARS-CoV-2.Naïve mice were immunized twice at day 0 and day 21 with eitherBNT162b2, with BA.1 or BA.4/5 S glycoprotein adapted monovalentvaccines, or the bivalent BNT162b2/BA.4/5 vaccine (FIG. 49(C)).Neutralizing titers against pseudoviruses expressing the wild-typestrain S glycoprotein, that of the previous VOCs Alpha or Delta, orOmicron BA.1, BA.2, BA.2.12.1, or BA.4/5 were determined in pVNT assaysusing sera drawn 14 days after the second immunization (d14D2). In serafrom BNT162b2 immunized mice, strong neutralizing activity against thewild-type strain was observed. In these sera, robust neutralizingactivity against Alpha and Delta was also detected (within a 4-foldrange of wild-type), whereas pVN₅₀ GMTs against Omicron variants weresubstantially (14 to 37-fold) lower than against wild-type (FIG. 52 ).Immunization with the Omicron BA.1 monovalent vaccine led to highneutralization of BA.1. In these sera, robust neutralization of OmicronBA.2 and BA.2.12.1 was also detected (within a 3-fold range of BA.1),while the pVN₅₀ GMTs against the wild-type strain and remaining VOCswere considerably (7 to 32-fold) lower than BA.1. Immunization with theOmicron BA.4/5 monovalent vaccine led to high neutralization of BA.4/5.In these sera, robust neutralization of Omicron BA.2 and BA.2.12.1 wasalso detected (within a 2.5-fold range of BA.4/5), while the pVN₅₀ GMTsagainst the wild-type strain and remaining VOCs were considerably (14to >42-fold lower than BA.4/5. Immunization with the BNT162b2/BA.4/5bivalent vaccine resulted in high neutralizing activity against BA.4/5.In contrast to the other vaccines, robust neutralizing activity againstthe wild-type strain and all remaining VOCs (within a 6-fold range ofBA.4/5) were also detected in these sera.

These results show that a monovalent vaccine in naïve animals (e.g.,animals that have not been previously administered against and/orinfected with SARS-CoV-2) can induce a high neutralizing antibodyresponse mostly in a variant-specific manner but can lose potency whentesting against more distant variants. In contrast, a bivalent vaccinecan induce strong and broad neutralizing antibody responses in naïveanimals.

Discussion

In the present Example, BA.4/BA.5 breakthrough infection of triple-mRNAvaccinated individuals is associated with robust neutralization of allcurrently or previously predominant Omicron subvariants, i.e.,pan-Omicron neutralization is observed in BA.4/5-breakthrough patients.These findings are consistent with a recent report showing strongcross-neutralization of Omicron BA.1, BA.2, as well as Beta and Delta insera from individuals vaccinated with BNT162b2 or an adenovirus-basedvaccine and subsequent BA.4 breakthrough infection (Ref. 27). In linewith those observations in humans, pan-Omicron neutralizing activity wasalso observed in the sera of mice that received an Omicron BA.4/5booster vaccine following primary immunization with BNT162b2, whereas anOmicron BA.1 boost induced strongly reduced neutralizing antibody titersagainst BA.4/BA.5. A bivalent BNT162b2/BA.4/5 boost elicited broadOmicron neutralization, albeit less pronounced than the BA.4/5monovalent booster. Together, these findings provide furtherunderstanding on how breakthrough infections or vaccine boosters adaptedto Omicron VOCs in a mono- or bivalent format shape immunity and suggestthat exposure to Omicron BA.4/5 S glycoprotein may confer heightenedprotection against the currently circulating and potential futureOmicron sublineage VOCs. The finding that immunization of naïve micewith the BNT162b2/BA.4/5 bivalent vaccine elicits strong neutralizingantibody responses against the wild-type strain as well as Omicron andnon-Omicron VOCs suggest that this bivalent approach can confer broadprotection to unvaccinated individuals not previously infectedSARS-CoV-2 (e.g., young pediatric patients), and thus may beparticularly suitable for these individuals.

While currently approved SARS-CoV-2 wild-type strain-based vaccines suchas BNT162b2 have proven effective at protecting against severe disease(Refs. 28-30), prevention of transmission remains a significantchallenge as new variants continue to emerge that are antigenicallydistant from the wild-type strain (Refs. 16-18, 20). The data describedin the present Example suggest that a mono- or bivalent BA.4/BA.5 Sglycoprotein adapted booster vaccine (e.g., a mono- or bivalent vaccinedescribed herein), can confer higher benefit against the highlyprevalent BA.4 and BA.5 VOCs than a vaccine based on a previouslydominant Omicron sublineage such as BA.1. Given their predominance inmany regions around the world and their high transmissibility (see,e.g., Refs., 8, 10, 11, and 13), it is possible that new variants withfurther growth advantage will emerge from Omicron BA.4 or BA.5 thatretain partial or full susceptibility to an Omicron BA.4/BA.5-adaptedvaccine. Boosting pre-existing immunity with an Omicron BA.4/5 Sglycoprotein-based adapted vaccine could therefore represent a suitablestrategy to address the current pandemic situation, while closemonitoring of virus evolution and epidemiological landscapes remainsinstrumental for guidance on potential further vaccine adaptations inresponse to emerging threats.

Materials and Methods

Human Study Design, Recruitment of Participants and Sample Collection

The objective of the study described in the present Example was toinvestigate the effect of Omicron BA.4/BA.5 breakthrough infection onthe cross-variant neutralization capacity of human sera. Neutralizingactivity was assessed in immune sera from triple-mRNA(BNT162b2/mRNA-1273)-vaccinated individuals with a confirmed subsequentSARS-CoV-2 breakthrough infection, which either occurred in a period ofOmicron BA.4/BA.5 lineage-dominance in Germany (mid-June to mid-July2022;) or was variant-confirmed (BA.4 or BA.5) by genome sequencing(mRNA-Vax³+BA.4/5) (FIG. 53 ). The neutralizing activity was compared tothat in immune sera from triple mRNA vaccinated individuals with aconfirmed subsequent SARS-CoV-2 breakthrough infection in a period ofOmicron BA.2 lineage-dominance (March to May 2022; mRNA-Vax³+BA.2), aperiod of Omicron BA.1 lineage-dominance in Germany (November 2021 tomid-January 2022; mRNA-Vax³+BA.1) (Refs. 1, 2), andtriple-BNT162b2-vaccinated individuals that were SARS-CoV-2-naïve(nucleocapsid seronegative) at the time of sample collection(BNT162b2³). Serum neutralizing capability was characterized usingpseudovirus and live SARS-CoV-2 neutralization assays. Data for thereference cohorts mRNA-Vax³+BA.2, mRNA-Vax³+BA.1, and BNT162b2³ werepreviously published (Refs. 16, 21). Participants from the mRNA-Vax³+OmiBA.4/5, mRNA-Vax³+Omi BA.2, and mRNA-Vax³+BA.1 cohorts were recruitedfrom University Hospital, Goethe University Frankfurt as part of anon-interventional study (protocol approved by the Ethics Board of theUniversity Hospital [No. 2021-560]) researching patients that hadexperienced Omicron breakthrough infection following vaccination forCOVID-19. Individuals from the BNT162b2³ cohort provided informedconsent as part of their participation in the Phase 2 trial BNT162-17(NCT05004181). The infections of 5 BA.4/5 and 4 BA.1 convalescentparticipants in this study were confirmed by genome sequencing (Ref.16).

All participants had no documented history of SARS-CoV-2 infection priorto vaccination. Participants were free of symptoms at the time of bloodcollection.

Serum was isolated by centrifugation of drawn blood at 2000×g for 10minutes and cryopreserved until use.

In Vitro Transcription and Lipid-Nanoparticle (LNP) Formulation of theRNA

The BNT162b2 vaccine was designed on a background of S sequences fromSARS-CoV-2 isolate Wuhan-Hu-1 (GenBank: MN908947.3) with pre-fusionconformation-stabilizing K986P and V987P mutations. Omicron BA.1 andOmicron BA.4/5 vaccine candidates were designed based on BNT162b2including sequence changes as shown in FIG. 54 . RNA production as wellas formulation were performed as described elsewhere (see, e.g., Ref. 32the contents of which are incorporated herein by reference in theirentirety). In brief, DNA templates were cloned into a plasmid vectorwith backbone sequence elements (T7 promoter, 5′ and 3′ UTR, 100nucleotide poly(A) tail) interrupted by a linker (A30LA70, 10nucleotides) for improved RNA stability and translational efficiency(see, e.g., Refs. 33, 34), amplified via PCR and purified. RNA was invitro transcribed by T7 RNA polymerase in the presence of atrinucleotide cap1 analogue ((m27,3′-O)Gppp(m2′-O)ApG; TriLink) and withN1-methylpseudouridine-5′-triphosphate (m1ψTP; Thermo Fisher Scientific)replacing uridine-5′-triphosphate (UTP) (Ref. 35). RNA was purifiedusing magnetic particles (Ref. 36) and RNA integrity was assessed bymicrofluidic capillary electrophoresis (Agilent 2100 Bioanalyzer) andall three RNAs show single sharp peaks resulting in comparable and highpurity as well as integrity (FIG. 55(A)). In addition, theconcentration, pH, osmolality, endotoxin level and bioburden of thesolution were determined.

One ionizable lipid((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)),two structural lipids (1,2-distearoyl-sn-glycero-3-phosphocholine [DSPC]and cholesterol) and one PEGylated lipid (2-[(polyethyleneglycol)-2000]-N,N-ditetradecylacetamide) were used for formulation ofRNA. After transfer into an aqueous buffer system via diafiltration, theLNP compositions were analyzed ensuring but not limited to high RNAintegrity and encapsulation efficacy, as well as a particle size below100 nm. The vaccine candidates were stored at −70 to −80° C. at aconcentration of 0.5 mg/mL till time point of usage.

In Vitro Expression of RNAs and Vaccines

HEK293T cells were transfected with 0.15 Vg BNT162b2 or Omicron-adaptedvaccines (lipid-nanoparticle-formulated), or with vaccine RNAs usingRiboJuice™ mRNA Transfection Kit (Merck Millipore) according to themanufacturer's instructions and incubated for 18 h. Transfected HEK293Tcells were stained with Fixable Viability Dye (eBioscience) andincubated with mFc-tagged recombinant human ACE-2 (Sino Biological). Asecondary donkey anti-mouse antibody conjugated with AF647 was used fordetection of surface expression. Cells were fixed (Fixation Buffer,Biolegend) prior to flow cytometry analysis using a FACSCelesta flowcytometer (BD Biosciences, BD FACSDiva software version 8.0.1) andFlowJo software version 10.6.2 (FlowJo, BD Biosciences).

Mouse Studies

All mouse studies were performed at BioNTech SE, and protocols wereapproved by the local authorities (local welfare committee) andconducted according to Federation of European Laboratory Animal ScienceAssociations recommendations. Study execution and housing were incompliance with the German Animal Welfare Act and Directive 2010/63/EU.Mice were kept in individually ventilated cages with a 12-h light/darkcycle, controlled environmental conditions (22±2° C., 45% to 65%relative humidity) and under specific-pathogen-free conditions. Food andwater were available ad libitum. Only mice with an unobjectionablehealth status were selected for testing procedures.

For immunization, female BALB/c mice (Janvier) (9-21 weeks old) wererandomly allocated to groups. BNT162b2 and Omicron-based vaccinescandidates were diluted in 0.9% NaCl and 1 μg of the vaccine candidatewas injected into the gastrocnemius muscle at a volume of 20 μl underisoflurane anesthesia. For the mouse booster study, mice were immunizedtwice (day 0 and 21) with BNT162b2. Third immunization with BNT162b2 andOmicron-based vaccines candidates occurred at day 104 after study startand mice were bled shortly before third immunization and as indicated inFIG. 49 . For the naïve moue study, animals were immunized at day 0 and21 with BNT162b2 and Omicron-based vaccines candidates. 14 days aftersecond immunization, blood was withdrawn. Peripheral blood was collectedfrom the vena facialis without anesthesia. Final bleeding was performedunder isoflurane anesthesia from the retro-orbital venous plexus. Forserum generation, blood was centrifuged for 5 min at 16,000 g and theserum was immediately used for downstream assays or stored at −20° C.till timepoint of usage.

VSV-SARS-CoV-2 S Variant Pseudovirus Generation

A recombinant replication-deficient vesicular stomatitis virus (VSV)vector that encodes green fluorescent protein (GFP) and luciferaseinstead of the VSV-glycoprotein (VSV-G) was pseudotyped with SARS-CoV-1S glycoprotein (UniProt Ref: P59594) and with SARS-CoV-2 S glycoproteinderived from either the wild-type strain (Wuhan-Hu-1, NCBI Ref:43740568), the Alpha variant (alterations: Δ69/70, Δ144, N501Y, A570D,D614G, P681H, T716I, S982A, D1118H), the Delta variant (alterations:T19R, G142D, E156G, Δ157/158, K417N, L452R, T478K, D614G, P681R, D950N),the Omicron BA.1 variant (alterations: A67V, Δ69/70, T95I, G142D,Δ143-145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N,N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H,T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K,L981F), the Omicron BA.2 variant (alterations: T19I, Δ24-26, A27S,G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N,N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y,N679K, P681H, N764K, D796Y, Q954H, N969K), the Omicron BA.2.12.1 variant(alterations: T19I, Δ24-26, A27S, G142D, V213G, G339D, S371F, S373P,S375F, T376A, D405N, R408S, K417N, N440K, L452Q, S477N, T478K, E484A,Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, S704L, N764K,D796Y, Q954H, N969K), and the Omicron BA.4/5 variant (alterations: T19I,Δ24-26, A27S, Δ69/70, G142D, V213G, G339D, S371F, S373P, S375F, T376A,D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R,N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K)according to published pseudotyping protocols (e.g., as described inRef. 3, the contents of which are incorporated by reference herein intheir entirety). A diagram of SARS-CoV-2 S glycoprotein alterations isshown in FIG. 32 and a separate alignment of S glycoprotein alterationsin Omicron sublineages is displayed in FIG. 33 .

In brief, HEK293T/17 monolayers (ATCC® CRL-11268™) cultured inDulbecco's modified Eagle's medium (DMEM) with GlutaMAX™ (Gibco)supplemented with 10% heat-inactivated fetal bovine serum (FBS[Sigma-Aldrich]) (referred to as medium) were transfected with Sangersequencing-verified SARS-CoV-1 or variant-specific SARS-CoV-2 Sexpression plasmid with Lipofectamine LTX (Life Technologies) followingthe manufacturer's instructions. At 24 hours after transfection, thecells were infected at a multiplicity of infection (MOI) of three withVSV-G complemented VSVΔG vector. After incubation for 2 hours at 37° C.with 7.5% CO₂, cells were washed twice with phosphate buffered saline(PBS) before medium supplemented with anti-VSV-G antibody (clone 8G5F11,Kerafast Inc.) was added to neutralize residual VSV-G-complemented inputvirus. VSV-SARS-CoV-2-S pseudotype-containing medium was harvested hoursafter inoculation, passed through a 0.2 μm filter (Nalgene) and storedat −80° C. The pseudovirus batches were titrated on Vero 76 cells (ATCC®CRL-1587™) cultured in medium. The relative luciferase units induced bya defined volume of a SARS-CoV-2 wild-type strain S glycoproteinpseudovirus reference batch previously described in Muik et al., 2021(Ref. 31), that corresponds to an infectious titer of 200 transducingunits (TU) per mL, was used as a comparator. Input volumes for theSARS-CoV-2 variant pseudovirus batches were calculated to normalize theinfectious titer based on the relative luciferase units relative to thereference.

Pseudovirus Neutralization Assay

Vero 76 cells were seeded in 96-well white, flat-bottom plates (ThermoScientific) at 40,000 cells/well in medium 4 hours prior to the assayand cultured at 37° C. with 7.5% CO₂. Human and mouse serum samples were2-fold serially diluted in medium with dilutions ranging from 1:5 to1:30,720 (human sera), from 1:40 to 1:102,400 for mouse booster study(mouse sera; starting dilution was 1:40 [pre-D3], 1:200 [d7D3]) as wellas 1:100 [d21D3, d35D3]) and in the naïve setting (mouse sera;monovalent vaccinated groups starting dilution 1:120 to 1:15,360 andbivalent vaccinated groups starting 1:100).VSV-SARS-CoV-2-S/VSV-SARS-CoV-1-S particles were diluted in medium toobtain 200 TU in the assay. Serum dilutions were mixed 1:1 withpseudovirus (n=2 technical replicates per serum per pseudovirus) for 30minutes at room temperature before being added to Vero 76 cellmonolayers and incubated at 37° C. with 7.5% CO₂ for 24 hours.Supernatants were removed and the cells were lysed with luciferasereagent (Promega). Luminescence was recorded on a CLARIOstar® Plusmicroplate reader (BMG Labtech), and neutralization titers werecalculated as the reciprocal of the highest serum dilution that stillresulted in 50% reduction in luminescence. Results for all pseudovirusneutralization experiments were expressed as geometric mean titers (GMT)of duplicates. If no neutralization was observed, an arbitrary titervalue of half of the limit of detection [LOD] was reported. Tables ofthe neutralization titers in human sera are provided.

Live SARS-CoV-2 Neutralization Assay

SARS-CoV-2 virus neutralization titers were determined by amicroneutralization assay based on cytopathic effect (CPE) at VisMederiS.r.l., Siena, Italy. In brief, human and mouse serum samples wereserially diluted 1:2 (n=2 technical replicates per serum per virus;starting at 1:10 for human samples and starting at 1:100 [post-boost,day 125] or at 1:50 [post-boost, day 139] for murine samples) andincubated for 1 hour at 37° C. with 100 TCID₅₀ of the livewild-type-like SARS-CoV-2 virus strain 2019-nCOV/ITALY-INMI1 (GenBank:MT066156), Omicron BA.1 strain hCoV-19/Belgium/rega-20174/2021(alterations: A67V, Δ69/70, T95I, G142D, Δ143-145, Δ211, L212I,ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N,T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y,N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F),sequence-verified Omicron BA.2 strain (alterations: T19I, Δ24-26, A27S,V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, S477N,T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H,R682W, N764K, D796Y, Q954H, N969K), or sequence-verified Omicron BA.4strain (alterations: V3G, T19I, Δ24-26, A27S, Δ69/70, G142D, V213G,G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452R,S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K,P681H, N764K, D796Y, Q954H, N969K) to allow any antigen-specificantibodies to bind to the virus. A diagram of S glycoprotein alterationsis shown in FIG. 48 . The 2019-nCOV/ITALY-INMI1 strain S glycoprotein isidentical in sequence to the wild-type SARS-CoV-2 S (Wuhan-Hu-1isolate). Vero E6 (ATCC® CRL-1586™) cell monolayers were inoculated withthe serum/virus mix in 96-well plates and incubated for 3 days(2019-nCOV/ITALY-INMI1 strain) or 4 days (Omicron BA.1, BA.2 and BA.4variant strain) to allow infection by non-neutralized virus. The plateswere observed under an inverted light microscope and the wells werescored as positive for SARS-CoV-2 infection (i.e., showing CPE) ornegative for SARS-CoV-2 infection (i.e., cells were alive without CPE).The neutralization titer was determined as the reciprocal of the highestserum dilution that protected more than 50% of cells from CPE andreported as GMT of duplicates. If no neutralization was observed, anarbitrary titer value of 5 (half of the LOD) was reported.

Statistical Analysis

The statistical method of aggregation used for the analysis of antibodytiters is the geometric mean and for the ratio of SARS-CoV-2 VOC titerand wild-type strain titer the geometric mean and the corresponding 95%confidence interval. The use of the geometric mean accounts for thenon-normal distribution of antibody titers, which span several orders ofmagnitude. The Friedman test with Dunn's correction for multiplecomparisons was used to conduct pairwise signed-rank tests of groupgeometric mean neutralizing antibody titers with a common control group.Spearman correlation was used to evaluate the monotonic relationshipbetween non-normally distributed datasets. All statistical analyses wereperformed using GraphPad Prism software version 9.

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Arora et al., Augmented neutralisation resistance of emerging    omicron subvariants BA.2.12.1, BA.4, and BA.5. Lancet Infect Dis,    (2022).-   20. Q. Wang et al., Antibody evasion by SARS-CoV-2 Omicron    subvariants BA.2.12.1, BA.4, & BA.5. Nature, (2022).-   21. A. Muik et al., Omicron BA.2 breakthrough infection enhances    cross-neutralization of BA.2.12.1 and BA.4/BA.5. bioRxiv, (2022).-   22. P. Du et al., A bivalent vaccine containing D614G and BA.1 spike    trimer proteins or a BA.1 spike trimer protein booster shows broad    neutralizing immunity. J Med Virol 94, 4287-4293 (2022).-   23. A. R. Branche et al., SARS-CoV-2 Variant Vaccine Boosters Trial:    Preliminary Analyses. medRxiv, (2022).-   24. A. Muik et al., Neutralization of SARS-CoV-2 lineage B.1.1.7    pseudovirus by BNT162b2 vaccine-elicited human sera. Science 371,    1152-1153 (2021).-   25. A. Muik et al., Neutralization of SARS-CoV-2 Omicron by BNT162b2    mRNA vaccine-elicited human sera. 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Example 19: Influence of BA.4/BA.5 Breakthrough Infections on OmicronImmune Responses

The present Example 19 is an extension of Examples 14, 17, and 18, anddescribes experiments in which serum samples collected from BA.1-,BA.2-, and BA.4/5-breakthrough cases were analyzed for neutralizationactivity against Omicron BA.4, BA.5, BA.4.6, BF.7, and BA.2.75 variants.In addition to confirming the results described in the previousExamples, the present Example 19 also provides further characterizationof antibody responses induced by BA.1-, BA.2-, and BA.4/5-breakthroughinfections in relation to BA.4.6, BF.7, and BA.2.75 variants. Thepresent Example further demonstrates that a BA.4/5 breakthroughinfection can produce a broader neutralization response than BA.1- andBA.2-breakthrough infections, with superior neutralization titersinduced against BA.4/5 and BA.4.6/BF.7 variants as compared to BA.1- andBA.2-breakthrough infections, and comparable neutralization titersagainst a BA.2.75 virus.

Continued evolution of the SARS-CoV-2 Omicron variant has led to theemergence of numerous sublineages with distinct patterns of neutralizingantibody evasion. The recently emerged sublineages BA.4.6, BF.7, andBA.2.75 have raised concerns as their prevalence slowly but steadilyincreases in many regions. The present Example investigated neutralizingactivity against these Omicron sublineages as well as the currentlydominant BA.4/BA.5 in immune sera of COVID-19 mRNA triple-vaccinatedindividuals with subsequent breakthrough infection with BA.1, BA.2, orBA.4/BA.5 SARS-CoV-2 variants. Sera from SARS-CoV-2 naïve triple- orquadruple mRNA-vaccinated individuals were also tested. Sera from BA.1,BA.2, and BA.4/BA.5 convalescents were broadly comparable in theirproficiency to cross-neutralize BA.2.75 relative to the SARS-CoV-2wild-type strain. The strongest cross-neutralization of OmicronBA.4.6/BF.7 was detected in BA.4/BA.5 convalescent sera. These findingsindicate that breakthrough infection with recent Omicron sublineages mayconfer at least partial protection against infection with newly emergingsublineages, and provides further evidence supporting the efficacy of aBA.4/5-specific vaccine (in particular, further evidence that aBA.4/5-specific vaccine can provide a broad immune response againstOmicron variants). The SARS-CoV-2 Omicron variant of concern (VOC)contains over 30 amino acid alterations in its spike (S) glycoprotein,which mediate partial escape from previously established immunity. As aresult, breakthrough infections with Omicron have been more frequentthan with previously circulating VOCs among vaccinated populations.Since Omicron's first emergence in November 2021, sublineages BA.1,BA.2, BA.4, and BA.5 consecutively dominated the pandemic landscape.While BA.5 has been the globally dominant sublineage since mid-2022,virus evolution continues to give rise to new sublineages that harboradditional amino acid alterations in their S glycoprotein. Descendantsof previous Omicron sublineages with slow but steady increases inprevalence have been reported in many countries across continents. Thesedescendants include Omicron BA.2.75, BA.4.6, and BF.7, which haveemerged from BA.2, BA.4, and BA.5, respectively.

Omicron BA.2.75 contains five amino acid alterations within theN-terminal domain (NTD) of the S protein that distinguishes it fromprevious Omicron Variants of Concern (VOCs), including its parentalsublineage BA.2 (FIG. 60 ). In addition, BA.2.75 has three alterationsin its receptor-binding domain (RBD) that are not found in BA.2, ofwhich G446S is shared with BA.1. The most prevalent strains of OmicronBA.4.6 and BF.7 have identical S glycoprotein sequences, which show highsimilarity with their respective parental lineages BA.4 and BA.5, whichalso share their S glycoprotein sequence. A single R346T alterationwithin the RBD differentiates BA.4.6/BF.7 from BA.4/BA.5, and abrogatesneutralization by the therapeutic monoclonal antibody (mAb) cilgavimab(Ref. 1). The combination of cilgavimab and tixagevimab (Evusheld™),which has high clinical relevance for pre-exposure COVID-19 prophylaxisin immunocompromised patients, is expected to be ineffective againstOmicron BA.4.6 and BF.7 given that tixagevimab lacks neutralizingactivity against Omicron BA.4/BA.5 and their descendants (Refs. 1 and2).

Primary SARS-CoV-2 immunity in many parts of the world is based oninfections with the original wild-type virus or wild-type strain-basedvaccines, such as the mRNA vaccines BNT162b2 and mRNA-1273. Immuneresponses are commonly further shaped through breakthrough infectionswith antigenically distinct Omicron sublineage viruses or recentlyauthorized Omicron-adapted vaccine boosters. An important question iswhether breakthrough infections and Omicron-adapted vaccine boosterselicit substantial neutralizing antibody responses against recentlyemerged Omicron sublineages currently gaining momentum.

The present Example investigated neutralizing activity against OmicronBA.4.6/BF.7 and BA.2.75 from immune sera isolated from five cohorts ofindividuals who received three or four doses of mRNA COVID-19 vaccines(BNT162b2/mRNA-1273 homologous or heterologous regimens), with orwithout subsequent breakthrough infections with Omicron sublineagevariants. Individual cohorts were BNT162b2 triple-vaccinatedSARS-CoV-2-naïve individuals (BNT162b2³; n=18), BNT162b2quadruple-vaccinated SARS-CoV-2-naïve elderly individuals (BNT162b2⁴;n=15), and triple mRNA-vaccinated individuals who experiencedbreakthrough infection with Omicron BA.1 (mRNA-Vax³+BA.1; n=14), BA.2(mRNA-Vax³+BA.2, n=19), or BA.4/BA.5 (mRNA-Vax³+BA.4/BA.5, n=17) (FIG.61 ). The study design includes cohorts that are thought to represent alarge proportion of the European and North American population, giventhe local health authorities' recommendation of a fourth vaccine dosefor elderly individuals and the high frequency of breakthroughinfections with Omicron compared to previous variants. Serumneutralizing activity was tested in a well-characterized pseudovirusneutralization test (pVNT) (Refs. 3-5) by determining 50% pseudovirusneutralization (pVN₅₀) geometric mean titers (GMTs) with pseudovirusesbearing the S glycoproteins of the SARS-CoV-2 wild-type strain orOmicron BA.4/BA.5 (BA.4 and BA.5 are identical in their S glycoproteinsequence), BA.4.6/BF.7 (BA.4.6 and BF.7 are identical in their Sglycoprotein sequence), or BA.2.75.

In the BNT162b2³, BNT162b2⁴, and mRNA-Vax³+BA.1 cohorts, pVN₅₀ titersagainst Omicron BA.4/BA.5 were significantly lower (GMTs reduced 5 to6-fold) than titers against the wild-type strain (FIG. 59(A)). Thereduction of BA.4/BA.5 neutralization in the mRNA-Vax³+BA.2 andmRNA-Vax³+BA.4/BA.5 cohorts was also statistically significant yet lesspronounced (GMTs 2 to 3-fold lower compared to wild-type) than in theother cohorts. pVN₅₀ titers against Omicron BA.4.6/BF.7 were furthersignificantly reduced compared to BA.4/BA.5 in mRNA-Vax³+BA.2 (GMT 239vs. 386; P<0.05). A considerable but statistically non-significantreduction in BA.4.6/BF.7 neutralization relative to BA.4/BA.5 was alsoseen in BNT162b2⁴ (GMT 55 vs. 121). For all other cohorts, OmicronBA.4.6/BF.7 GMTs were largely comparable to those against OmicronBA.4/BA.5 and titers in the mRNA-Vax³+BA.4/BA.5 were the most robust(GMT 443). In mRNA-Vax³+BA.4/BA.5, titers against BA.2.75 were reduced1.8-fold compared to BA.4/BA.5 (GMT 295 vs. 521), while neutralizingGMTs against BA.2.75 were even increased 2-fold compared to thoseagainst BA.4/BA.5 in mRNA-Vax³+BA.1 (GMT 525 vs. 263). Titers againstBA.2.75 were comparable to those against BA.4/5 in the other cohorts.

To allow for assessment of neutralization breadth irrespective of themagnitude of antibody titers, the Omicron sublineage pVN₅₀ GMTs werenormalized against the wild-type strain. GMT ratios for all Omicronsubvariant pseudoviruses were comparable in the BNT162b2³ and BNT162b2⁴cohorts (ratios ≤0.22 for all pseudoviruses, FIG. 59(B)), indicatingthat a fourth dose of BNT162b2 did not improve cross-neutralization ofthe tested subvariants despite a slight overall increase in antibodytiters. Cross-neutralization of BA.4/BA.5 and BA.4.6/BF.7 weresignificantly (p<0.05) stronger in sera from mRNA-Vax³+BA.2 compared toBNT162b2³ (GMT ratios 0.37 vs 0.17 for BA.4/BA.5, and 0.23 vs 0.12 forBA.4.6/BF.7). The GMT ratios were even higher in the mRNA-Vax³+BA.4/BA.5cohort for both the BA.4/BA.5 pseudovirus (GMT ratio 0.48, p<0.01 versusBNT162b2³) and the BA.4.6/BF.7 pseudovirus (0.41, p<0.0001).Cross-neutralization of the Omicron BA.2.75 pseudovirus was broadlycomparable in most cohorts, with a significant (p<0.05) increase seenonly in mRNA-Vax³+BA.1 compared to BNT162b2³.

The finding that neutralizing activity against Omicron BA.4.6/BF.7 isfurther reduced compared to BA.4/BA.5 in sera of vaccinated individualsand BA.2 convalescents suggests that the R346T alteration mediatesfurther escape from neutralizing antibodies in polyclonal sera.Convergent evolution of the RBD at this site in Omicron BA.4.6, BF.7,and additional currently less prevalent strains (Ref. 1) indicates thatthe resulting immune escape may confer considerable growth advantage.The findings summarized here indicate that Omicron BA.4/BA.5breakthrough infection refocuses neutralizing antibody responses topartially restore BA.4.6/BF.7 neutralization. A similarcross-neutralization pattern of the Omicron lineage in BA.4/BA.5breakthrough infected humans and in mice boosted with an OmicronBA.4/5-adapted vaccine has also been observed, as demonstrated inExample 18. Hence, such findings provide evidence supporting thatOmicron BA.4/5-adapted vaccine boosters can also elicit relevantneutralizing antibody responses against BA.4.6/BF.7 in humans.

The observation that cross-neutralization of Omicron BA.2.75 is broadlycomparable to that of BA.4/5 is consistent with previous reports (Refs.2, 6, and 7). Without wishing to be bound by a particular theory, thisobservation suggests that the growth advantage of BA.2.75 over BA.5 maybe related to factors other than immune evasion. For example, in someembodiments, minor differences in the susceptibility of Omicron BA.2.75and BA.4/BA.5 to neutralization by BA.1 and BA.4/BA.5 convalescent seramay point towards amino acid alterations with a potentialcontext-dependent role in immune evasion.

The investigation summarized in the present Example focused onneutralization of the new Omicron sublineages BA.4.6/BF.7, and BA.2.75,as these sublineages could potentially displace BA.5 in the futureaccording to their current trajectory. The finding that Omicronbreakthrough infection is associated with enhanced cross-neutralizationof the new sublineages is consistent with the data provided in theprevious Examples, showing enhanced neutralization breadth, includingagainst earlier Omicron sublineages and previous SARS-CoV-2 VOCs.Together these findings show that Omicron BA.4/BA.5 breakthroughinfection is associated with the broadest neutralization against allvariants, including Omicron sublineages, providing evidence supportingthat vaccination with RNA encoding a SARS-CoV-2 S protein comprisingmutations characteristic of the BA.4/5 variant can produce a broadneutralization response.

Materials and Methods

Study Design, Recruitment of Participants and Sample Collection

The objective of this study was to investigate the cross-neutralizingactivity of five different panels of sera against Omicron BA.4.6/BF.7and BA.2.75 sub-lineages compared to SARS-CoV-2 wild-type and OmicronBA.4/BA.5. Neutralizing activity was assessed in immune sera from (i)SARS-CoV-2-naïve triple-BNT162b2-vaccinated younger adult individuals(BNT162b2³), (ii) SARS-CoV-2-naïve quadruple-BNT162b2-vaccinated olderadult individuals (BNT162b2⁴), and triple-mRNA(BNT162b2/mRNA-1273)-vaccinated individuals with a confirmed subsequentSARS-CoV-2 breakthrough infection which either occurred (iii) in aperiod of Omicron BA.1 lineage-dominance (November 2021 to mid-January2022; mRNA-Vax³+BA.1), (iv) in a period of Omicron BA.2lineage-dominance (March to May 2022; mRNA-Vax³+BA.2), or (v) in aperiod of Omicron BA.4/BA.5 lineage-dominance in Germany (mid-June tomid-July 2022; mRNA-Vax³+BA.4/5). Serum neutralizing capability wascharacterized using pseudovirus neutralization assays. SARS-CoV-2wild-type and Omicron BA.4/BA.5 neutralization data for cohortsBNT162b2³, mRNA-Vax³+BA.1, mRNA-Vax³+BA.2, mRNA-Vax³+BA.4/5 werepreviously published.

Participants from the mRNA-Vax³+Omi BA.1, mRNA-Vax³+Omi BA.2, andmRNA-Vax³+BA.4/5 cohorts were recruited from University Hospital, GoetheUniversity Frankfurt as part of a non-interventional study (protocolapproved by the Ethics Board of the University Hospital [No. 2021-560])researching patients that had experienced Omicron breakthrough infectionfollowing vaccination for COVID-19. Individuals from the BNT162b2³ andBNT162b2⁴ cohort provided informed consent as part of theirparticipation in the Phase 2 trial BNT162-17 (NCT05004181) and BNT162-16Substudy F (NCT04955626), respectively. All participants had nodocumented history of SARS-CoV-2 infection prior to vaccination.Participants were free of symptoms at the time of blood collection.

Serum was isolated by centrifugation of drawn blood at 2000×g for 10minutes and cryopreserved until use.

A recombinant replication-deficient vesicular stomatitis virus (VSV)vector that encodes green fluorescent protein (GFP) and luciferaseinstead of the VSV-glycoprotein (VSV-G) was pseudotyped with SARS-CoV-1S glycoprotein (UniProt Ref: P59594) or with SARS-CoV-2 S glycoproteinderived from either the wild-type strain (Wuhan-Hu-1, NCBI Ref:43740568), the Omicron BA.4/BA.5 variant (alterations: T19I, Δ24-26,A27S, Δ69/70, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N,R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y,Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K), theOmicron BA.4.6/BF.7 variant (alterations: T19I, Δ24-26, A27S, Δ69/70,G142D, V213G, G339D, R346T, S371F, S373P, S375F, T376A, D405N, R408S,K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H,D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K), and the OmicronBA.2.75 variant (alterations: T19I, Δ24-26, A27S, G142D, K147E, W152R,F157L, 1210V, V213G, G257S, G339H, S371F, S373P, S375F, T376A, D405N,R408S, K417N, N440K, G446S, N460K, S477N, T478K, E484A, Q498R, N501Y,Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K) accordingto published pseudotyping protocols (Berger Rentsch, Marianne, and GertZimmer. “A vesicular stomatitis virus replicon-based bioassay for therapid and sensitive determination of multi-species type I interferon.”PloS one 6.10 (2011): e25858). A diagram of SARS-CoV-2 S glycoproteinalterations is shown in FIG. 62 and a separate alignment of Sglycoprotein alterations in Omicron VOCs is displayed in FIG. 60 .

In brief, HEK293T/17 monolayers (ATCC® CRL-11268™) cultured inDulbecco's modified Eagle's medium (DMEM) with GlutaMAX™ (Gibco)supplemented with 10% heat-inactivated fetal bovine serum (FBS[Sigma-Aldrich]) (referred to as medium) were transfected with Sangersequencing-verified variant-specific SARS-CoV-2 S expression plasmidwith Lipofectamine LTX (Life Technologies) following the manufacturer'sinstructions. At 24 hours after transfection, the cells were infected ata multiplicity of infection (MOI) of three with VSV-G complemented VSVΔGvector. After incubation for 2 hours at 37° C. with 7.5% CO₂, cells werewashed twice with phosphate buffered saline (PBS) before mediumsupplemented with anti-VSV-G antibody (clone 8G5F11, Kerafast Inc.) wasadded to neutralize residual VSV-G-complemented input virus.VSV-SARS-CoV-2-S pseudotype-containing medium was harvested 20 hoursafter inoculation, passed through a 0.2 μm filter (Nalgene) and storedat −80° C. The pseudovirus batches were titrated on Vero 76 cells (ATCC®CRL-1587™) cultured in medium. The relative luciferase units induced bya defined volume of a SARS-CoV-2 wild-type strain S glycoproteinpseudovirus reference batch previously described in Muik et al., 2021(Muik, Alexander, et al. “Neutralization of SARS-CoV-2 lineage B. 1.1. 7pseudovirus by BNT162b2 vaccine-elicited human sera.” Science 371.6534(2021): 1152-1153), that corresponds to an infectious titer of 200transducing units (TU) per mL, was used as a comparator. Input volumesfor the SARS-CoV-2 variant pseudovirus batches were calculated tonormalize the infectious titer based on the relative luciferase unitsrelative to the reference.

Pseudovirus Neutralization Assay

Vero 76 cells were seeded in 96-well white, flat-bottom plates (ThermoScientific) at 40,000 cells/well in medium 4 hours prior to the assayand cultured at 37° C. with 7.5% CO2. Human serum samples were 2-foldserially diluted in medium with dilutions ranging from 1:10 to 1:10,240.VSV-SARS-CoV-2-S particles were diluted in medium to obtain 200 TU inthe assay. Serum dilutions were mixed 1:1 with pseudovirus (n=2technical replicates per serum per pseudovirus) for 30 minutes at roomtemperature before being added to Vero 76 cell monolayers and incubatedat 37° C. with 7.5% CO₂ for 24 hours. Supernatants were removed and thecells were lysed with luciferase reagent (Promega). Luminescence wasrecorded on a CLARIOstar® Plus microplate reader (BMG Labtech), andneutralization titers were calculated as the reciprocal of the highestserum dilution that still resulted in 50% reduction in luminescence.Results for all pseudovirus neutralization experiments were expressed asgeometric mean titers (GMT) of duplicates. If no neutralization wasobserved, an arbitrary titer value of half of the limit of detection[LOD] was reported.

Statistical Analysis

The statistical method of aggregation used for analysis of antibodytiters was the geometric mean and for the ratio of SARS-CoV-2 VOC titerand wild-type strain titer the geometric mean and the corresponding 95%confidence interval. The use of the geometric mean accounts for thenon-normal distribution of antibody titers, which span several orders ofmagnitude. The Friedman test with Dunn's correction for multiplecomparisons was used to conduct pairwise signed-rank tests of groupgeometric mean neutralizing antibody titers with a common control group.The Kruskal-Wallis test with Dunn's correction for multiple comparisonswas used to conduct unpaired signed-rank tests of group GMT ratios. Allstatistical analyses were performed using GraphPad Prism softwareversion 9.

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Example 20: Clinical Trial Results Confirming that a Booster Dose thatIncludes Omicron BA.4/5-Specific Vaccine can Induce a Strong ImmuneResponse Against Omicron BA.4/5 Variants

The present Example provides clinical trial data confirming that aBA.4/5-specific vaccine (e.g., a bivalent RNA vaccine comprising a firstRNA encoding a SARS-CoV-2 S protein of a Wuhan strain and a second RNAencoding a SARS-CoV-2 S protein comprising one or more mutationscharacteristic of a BA.4/5 Omicron variant as described herein) caninduce a strong immune response in subjects. Specifically, the presentExample provides data demonstrating that such an RNA vaccine can inducean immune response (e.g., neutralization antibody titers) that arehigher against a BA.4/5 variant than an immune response induced by anRNA vaccine encoding a SARS-CoV-2 S protein of a Wuhan strain. Theseresults confirm the insights provided by the BA.4/5-breakthroughinfection data and mouse experiment data described in the previousExamples, with respect to the benefits of administering aBA.4/5-specific vaccine (e.g., ones described herein).

The present Example provides immune response data from a Phase 2/3clinical trial in which a 30-μg booster dose of an OmicronBA.4/BA.5-adapted bivalent COVID-19 vaccine (comprising 15 μg of RNAencoding a full length SARS-CoV-2 S protein of a Wuhan variant, forexample, in some embodiments an RNA comprising a nucleotide sequencethat is at least 95% or higher (including and up to 100%) identical toSEQ ID NO: 20 (e.g., in some embodiments BNT 162b2), and 15 μg of RNAencoding a full length SARS-CoV-2 S protein comprising one or moremutations characteristic of an Omicron BA.4/BA.5, for example, in someembodiments, an RNA comprising a nucleotide sequence that is at least95% or higher (including and up to 100%) identical to SEQ ID NO:72) wasadministered to individuals 18 to 55 years of age (n=38) and those olderthan 55 years of age (n=36) who had previously received three doses ofan RNA vaccine encoding a SARS-CoV-2 S protein of a Wuhan strain (e.g.,BNT162b2). A comparator group of participants older than 55 years of age(n=40) were administered a 30-pg booster dose (as a fourth dose) of anRNA vaccine encoding a SARS-CoV-2 S protein of a Wuhan strain. In thepresent Example, subjects who were administered a bivalent vaccine asdescribed herein received their last dose of the vaccine that encodes aSARS-CoV-2 S protein of a Wuhan strain on an average of 11 months priorto administration of a bivalent vaccine, whereas subjects who wereadministered an RNA vaccine encoding a SARS-CoV-2 protein of a Wuhanstrain, e.g., BNT162b2 (as a fourth dose) received their third dose onan average of 6 months previously. Despite this difference, pre-boosterantibody titers were similar in both groups. Both groups includedsubjects with evidence of prior SARS-CoV-2 infection and subjectswithout prior SARS-CoV-2 infection.

Among subjects administered an Omicron BA.4/BA.5-specific bivalentvaccine, a substantially higher increase in OmicronBA.4/BA.5-neutralizing antibody titers was observed as compared topre-booster levels. For individuals 18 to 55 years of age administered abivalent vaccine, the geometric mean titer (GMT) against OmicronBA.4/BA.5 was about 600 (e.g., 606), which was a 9.5-fold rise (95% CI:6.7, 13.6) from pre-booster levels. For individuals older than 55 yearsadministered a BA.4/5 bivalent vaccine, the GMT was about 900 (e.g.,896), which was a 13.2-fold rise (95% CI: 8.0, 21.6) from pre-boosterlevels. By contrast, participants over 55 years of age who received a30-μg booster dose of an RNA vaccine encoding a SARS-CoV-2 S protein ofa Wuhan strain, a lower neutralizing antibody response was observedagainst Omicron BA.4/BA.5 measured 1-month post booster. For theseparticipants, the GMT was about 230 (e.g., 236), representing a 2.9-foldrise (95% CI: 2.1, 3.9) from pre-booster levels. The safety profileobserved in the clinical trial for the bivalent vaccine was favorable,and consistent with BNT162b2.

In addition, when examining populations of subjects with and withoutevidence of a prior SARS-CoV-2 infection, an increase in neutralizingantibodies against Omicron BA.4/BA.5 following a booster dose of abivalent vaccine was observed in both populations, demonstrating that aBA.4/5-bivalent vaccine can improve protection in subjects that receivesuch a vaccine, regardless of prior infection status.

Example 21: Improved Neutralization of Omicron BA.4/5, BA.4.6,BA.2.75.2, BQ.1.1, and XBB.1 with a Bivalent BA.4/5 Vaccine

The present Example provides further data demonstrating that a bivalentRNA vaccine comprising RNA encoding a SARS-CoV-2 S protein of a Wuhanstrain and RNA encoding a SARS-CoV-2 S protein comprising one or moremutations characteristic of a BA.4/5 Omicron variant (e.g., a bivalentvaccine described herein) can provide an improved immune response ascompared to a monovalent RNA vaccine encoding a SARS-CoV-2 S protein ofa Wuhan strain. For example, in some embodiments, such an improvedimmune response includes increased neutralization titers against one ormore, including, two or more, three or more, four or more, five or more,six or more, SARS-CoV-2 variants of concern. In some embodiments, suchan improved immune response includes increased neutralization titersagainst one or more, including, two or more, three or more, four ormore, five or more, six or more, Omicron variants of concern. In someembodiments, such an improved immune response includes increasedneutralization titers against a larger number of Omicron variants ofconcern, as compared to that observed with a monovalent RNA vaccineencoding a SARS-CoV-2 S protein of a Wuhan strain.

In particular, the present Example provides data demonstrating that sucha bivalent vaccine can induce an improved immune response (e.g., in someembodiments higher neutralizing responses) against BA.5-derivedsublineages (e.g., BA.4.6, BQ.1.1, and XBB.1) and BA.2-derivedsublineages (e.g., BA.2.75.2) as compared to a monovalent RNA vaccineencoding a SARS-CoV-2 S protein of a Wuhan strain when administered as a4th dose booster. The present Example also provides data demonstratingthat subjects with SARS-CoV-2 infection history (e.g., subjects withprevious or current SARS-CoV-2 infection) can develop higher immuneresponses (e.g., higher neutralizing titers) after administration of a4^(th) booster dose (e.g., of a monovalent or a bivalent vaccine)relative to subjects who have not been infected with SARS-COV-2. Thepresent Example demonstrates that a BA.4/5 bivalent vaccine can inducean improved immune response (e.g., improved neutralizing response) ascompared to a monovalent RNA vaccine encoding a SARS-CoV-2 S protein ofa Wuhan strain regardless of SARS-CoV-2 infection history. The presentExample also provides data demonstrating that improvements in immuneresponse against certain Omicron sublineages (e.g., Omicron sublineagestested herein) provided by a BA.4/5 bivalent vaccine relative to a Wuhanmonovalent vaccine are greater for subjects who have not been previouslyinfected with SARS-CoV-2 as compared to subjects who have beenpreviously infected with SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicronvariant has continued to evolve globally into many sublineages since itsemergence in November 2021.

To mitigate the ongoing Omicron pandemic, the U.S. FDA authorizedemergency use of a bivalent BA.4/5-vaccine in September 2022. The U.S.FDA authorized bivalent vaccine contains two mRNAs: one encodingoriginal (Wuhan) SARS-CoV-2 Spike protein (BNT162b2) and anotherencoding a SARS-CoV-2 S protein comprising mutations characteristic ofan Omicron BA.4/5 variant (comprising in some embodiments an amino acidsequence of SEQ ID NO: 69, and in some embodiments SEQ ID NO: 72). SinceU.S. FDA approval, new Omicron BA.2- and BA.5-descendent sublineages(e.g., BA.4.6, BA.2.75.2, BQ.1.1, and XBB.1) have emerged and becomeprevalent(https://covid.cdc.gov/covid-data-tracker/#variant-proportions).Although early epidemiological data suggest no increase in diseaseseverity, these new sublineages have accumulated additional spikemutations that could further evade vaccine- and/or infection-elicitedantibody neutralization (Refs. 2-4).

The present Example describes data from a clinical trial in whichneutralization titers against Omicron sublineages BA.4/5, BA.4.6,BA.2.75.2, BQ.1.1, and XBB.1 were measured in sera collected fromsubjects administered as a fourth dose (a) a 30-μg booster dose of anOmicron BA.4/BA.5-adapted bivalent COVID-19 vaccine, which in someembodiments comprises 15 μg of RNA comprising a nucleotide sequence thatis at least 95% (including and up to 100%) identical to SEQ ID NO: 20,and 15 μg of RNA encoding a full length SARS-CoV-2 S protein comprisingone or more mutations characteristic of an Omicron BA.4/BA.5 variant andcomprising a nucleotide sequence that is at least 95% (including and upto 100%) identical to SEQ ID NO:72. In the present Example, an OmicronBA.4/BA.5-adapted bivalent COVID-19 vaccine comprising (i) 15 μg of RNAcomprising the sequence of SEQ ID NO: 20 and (ii) 15 μg of RNAcomprising the sequence of SEQ ID NO: 72 was administered to the testedsubjects.

Participants >55-years-old who had previously received 3 doses of avaccine that delivered a SARS-CoV-2 S protein of a Wuhan strain (in thepresent Example, subjects had previously received three 30-μg doses ofBNT162b2) were administered a 4th booster dose comprising 30-μg ofmonovalent BNT162b2 at ˜6.6-months-post-dose-3 or 30-μg of a bivalentvaccine (15-μg BNT162b2 plus 15-μg BA.4/5) at ˜11-months-post-dose-3.Serum was collected on the day dose 4 was administered (Pre serumsamples) and at 1-month-post-dose-4 (1MPD4 serum samples). Allparticipants were screened for evidence of previous and currentSARS-CoV-2 infection by viral nucleocapsid antibodies and RT-PCR tests;both vaccine groups in the neutralization analysis were equallydistributed among those with or without evidence of infection. Forneutralization testing, the complete spike gene from Omicron BA.4/5(BA.4 and BA.5 encode an identical spike sequence), BA.2.75.2, BQ.1.1,or XBB.1 was cloned into the backbone of fluorescent reporterUSA-WA1/2020 SARS-CoV-2 (a strain isolated in January 2020, see Ref. 5).The resulting wild-type—(WT), BA.4/5-, BA.2.75.2-, BQ.1.1-, andXBB.1-spike mNG USA-WA1/2020 were used to measure 50% fluorescent focusreduction neutralization titers (FFRNT₅₀) for each serum samplecollected.

For all participants (including subjects with and without evidence ofSARS-CoV-2 infection), a 4^(th) dose of monovalent BNT162b2 vaccine wasfound to induce a 3.0×, 2.9×, 2.3×, 2.1×, 1.9×, and 1.5× geometric meanneutralizing titer fold rise (GMFR) against Wuhan, BA.4/5, BA.4.6,BA.2.75.2, BQ.1.1, and XBB.1, respectively; a bivalent vaccine was foundto induce 5.8×, 13.0×, 11.1×, 6.7×, 8.7×, and 4.8×GMFRs (FIG. 63(A)).For individuals without SARS-CoV-2 infection history, BNT162b2 induced4.4×, 3.0×, 2.6×, 2.1×, 1.5×, and 1.3×GMFRs, respectively; the bivalentvaccine induced 9.9×, 25.9×, 21.2×, 8.6×, 13.0×, and 4.6×GMFRs (FIG.63(B)). For individuals with previous SARS-CoV-2 infection, BNT162b2induced 2.0×, 2.8×, 2.1×, 2.1×, 2.2×, and 1.8×GMFRs, respectively; thebivalent vaccine induced 3.5×, 6.7×, 6.2×, 5.3×, 6.3×, and 4.9×GMFRs(FIG. 63C). Despite different intervals between doses 3 and 4,pre-dose-4 neutralizing titers were similar in the monovalent andbivalent vaccine groups in the all participants and the withoutinfection groups.

The present Example provides at least three findings. First, a bivalentBA.4/5 vaccine consistently elicited higher neutralizing responsesagainst BA.5-derived sublineages (BA.4.6, BQ.1.1, and XBB.1) andBA.2-derived sublineage (BA.2.75.2) than a monovalent RNA vaccineencoding a SARS-CoV-2 S protein when administered as a 4th dose booster.Second, individuals with evidence of previous or current SARS-CoV-2infection developed higher neutralizing titers than those withoutinfection after a 4th dose booster. The improved neutralizing responsesinduced by a bivalent vaccine was observed regardless of SARS-CoV-2infection history. Third, for each tested Omicron sublineage, thedifference between BNT162b2- and bivalent-GMFR was greater for serawithout previous infection than those with previous infection.

Among all Omicron sublineages, BA.2.75.2, BQ.1.1, and XBB.1 exhibitedthe greatest evasion of vaccine-elicited neutralization; however,neutralizing titers following a bivalent booster were several foldhigher than those following a monovalent RNA encoding a SARS-CoV-2 Sprotein of a Wuhan strain. These data demonstrate that a bivalentvaccine is more immunogenic against circulating Omicron sublineages andunderscore the importance of monitoring real-world effectiveness.

Methods

Cells

Vero E6 (ATCC® CRL-1586) purchased from the American Type CultureCollection (ATCC, Bethesda, MD) and Vero E6 cells expressing TMPRSS2purchased from SEKISUI XenoTech, LLC were maintained in a high-glucoseDulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovineserum (FBS; HyClone Laboratories, South Logan, UT) and 1%penicillin/streptomycin at 37° C. with 5% C02. Culture media andantibiotics were purchased from Thermo Fisher Scientific (Waltham, MA).The cell line was tested negative for Mycoplasma.

Human Serum

In participants >55 years of age who previously received three 30-μgBNT162b2 doses, serum samples were collected just prior to and 1 monthpost-boost with a 4th dose booster of monovalent original 30-μg BNT162b2or 30-μg bivalent BA.4/BA.5 vaccine (15 μg original with 15 μgBA.4/BA.5). The protocol and informed consent were approved byinstitutional review boards for each of the investigational centersparticipating in the study. The study was conducted in compliance withall International Council for Harmonisation Good Clinical Practiceguidelines and the ethical principles of the Declaration of Helsinki.The median time between dose-3 and -4 was 6.3 and 11.3 months forBNT162b2 and the bivalent BA.4/5 vaccine, respectively. All participantswere screened by SARS-CoV-2 nucleocapsid Ig serological test forpreexisting SARS-CoV-2 or RT-PCR for existing infection. The subset ofparticipant's sera (approximately 40 per vaccine group) selected forneutralization testing were equally distributed between those with andwithout evidence of infection by either test. Human sera wereheat-inactivated at 56° C. for 30 min before the neutralization test.

Recombinant Omicron Sublineages-FP SARS CoV-2 Viruses

Recombinant Omicron sublineage BA.4/5-, BA.4.6-, BA.2.75.2-, BQ.1.1-,and XBB.1-spike fluorescent protein (FP) SARS-CoV-2s was constructed byengineering the complete spike gene from the indicated variants into aninfectious cDNA clone of mNG USA-WA1/2020 and reported previously (Refs.6-10). Viruses were rescued post 2-3 days after electroporation andserved as P0 stock. P0 stock was further passaged once on Vero E6 cellsto produce P1 stock. The spike gene was sequenced from all P1 stockviruses to ensure no undesired mutation. The infectious titer of the P1virus was quantified by fluorescent focus assay on Vero E6 cells. The P1virus was used for the neutralization test.

Fluorescent Focus Reduction Neutralization Test (FFRNT)

Neutralization titers of human sera were measured by FFRNT using theUSA-WA1/2020-, BA.4/5, BA.4.6-, BA.2.75.2-, BQ.1.1- and XBB.1-spike FPSARS-CoV-2s. All sera were tested sequentially, USA-WA1/2020 and BA.4/5followed by the remaining Omicron sublineages. The details of the FFRNTprotocol were reported previously (see Refs. 6 and 10-13). Briefly,2.5×104 Vero E6 cells per well were seeded in 96-well plates (GreinerBio-One™). The cells were incubated overnight. On the next day, eachserum was 2-fold serially diluted in the culture medium with the firstdilution of 1:20 (final dilution range of 1:20 to 1:20,480). The dilutedserum was incubated with 100-150 FFUs of FP SARS-CoV-2 at 37° C. for 1h, after which the serum virus mixtures were loaded onto the pre-seededVero E6 cell monolayer in 96-well plates. After 1 h infection, theinoculum was removed and 100 μl of overlay medium (supplemented with0.8% methylcellulose) was added to each well. After incubating theplates at 37° C. for 16 h, raw images of FP foci were acquired usingCytation™ 7 (BioTek) armed with 2.5× FL Zeiss objective with awide-field of view and processed using the Gene 5 software settings (GFP[469,525] threshold 4000, object selection size 50-1000 μm). The foci ineach well were counted and normalized to the non-serum-treated controlsto calculate the relative infectivities. The FFRNT₅₀ value was definedas the minimal serum dilution that suppressed >50% of fluorescent foci.The neutralization titer of each serum was determined in duplicateassays, and the geometric mean was taken. All attempts at replicationwere successful. Data were initially plotted in GraphPad Prism 9software and assembled in Adobe Illustrator.

REFERENCES CITED IN EXAMPLE 21 (EACH INCORPORATED BY REFERENCE HEREIN INITS ENTIRETY)

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Example 22: Distinct Cross-Neutralization of Omicron Sublineages byVaccine-Elicited and Convalescent Immune Sera

Serum samples were drawn from (i) SARS-CoV-2-naïve individualstriple-vaccinated with BNT162b2 or (ii) quadruple-vaccinated withBNT162b2, and individuals with three doses of mRNA COVID-19 vaccine(BNT162b2/mRNA-1273 homologous or heterologous regimens) whosubsequently had a breakthrough infection with (iii) Omicron BA.1, (iv)Omicron BA.2 or (v) Omicron BA.4/BA.5. Breakthrough infections occurredat a time of respective variant of concern dominance (BA.1: November2021 to January 2021, BA.2: March to May 2022, BA.4/5: mid-June tomid-July 2022) and/or were variant confirmed by genome sequencing.Neutralization titers were measured using a pseudovirus neutralizationassay, using pseudoviruses bearing the SARS-CoV-2 S protein of a BA.4/5,BA.4.6/BF.7, BQ.1.1, BA.2.75, BA.2.75.2, or XBB Omicron variants on thesurface (e.g., using methods described in the previous Examples; FIG. 65). 50% pseudovirus neutralization titers (pVNT₅₀) are shown in FIG.64(A). Neutralization breadth irrespective of the magnitude of antibodytiters was assessed by normalizing the Omicron sublineage pVNT₅₀ againstthose for the wild-type strain (FIG. 64(B)). In thetriple-/quadruple-vaccinated individuals without breakthrough infection,pVN₅₀ GMTs against Omicron BA.4/5 were 5 to 6-fold lower than GMTsagainst the wild-type strain (GMTs against BA.4/5 in the range of69-121) (FIG. 64(A)). GMTs against BA.4/5 were similarly reduced in BA.1convalescents (GMT 263, 5-fold lower than wild-type), whereas in theBA.2 and BA.4/BA.5 convalescent cohorts titers against BA.4/5 remainedhigher (GMTs of 386 and 521, respectively; 3 and 2-fold lower thanwild-type).

In all three convalescent cohorts, neutralizing titers against OmicronBA.4.6/BF.7 and BA.2.75 were robustly above those oftriple-/quadruple-vaccinated SARS-CoV-2 naïve individuals (GMT range239-525 for convalescents as compared to 55-139 for naïves). In theconvalescents, Omicron BA.4.6/BF.7 and BA.2.75 GMTs were largelycomparable with no significant differences to those against OmicronBA.4/BA.5. In contrast, pVN₅₀ titers against Omicron BQ.1.1, BA.2.75.2,and XBB were lower than those against BA.4/5 across cohorts. Titersagainst BQ.1.1 were overall low in the SARS-CoV-2 naïve vaccinatedcohorts and BA.1 convalescents (GMTs 538), and higher in the BA.2 andBA.4/BA.5 convalescent cohorts (GMTs 100 and 154, respectively). Titersagainst BA.2.75.2 and XBB were relatively low across cohorts (GMTs 588and ≤33, respectively).

To assess neutralization breadth irrespective of the magnitude ofantibody titers, Omicron sublineage pVN₅₀ GMTs were normalized againstthose for the wild-type strain. GMT ratios for all Omicron subvariantpseudoviruses were comparable in the BNT162b2³ and BNT162b2⁴ cohorts(FIG. 64(B)), indicating that a fourth dose did not improvecross-neutralization of the tested sublineages. GMT ratios were in therange of 0.09-0.22 for BA.4/5, BA.4.6/BF.7, and BA.2.75 and ≤0.05 forBQ.1.1, BA.2.75.2, and XBB in both cohorts.

Cross-neutralization of BA.4/BA.5 and BA.4.6/BF.7 was significantly(p<0.05) higher in sera from BA.2.convalescents as compared totriple-vaccinated individuals (GMT ratios 0.37 vs 0.17 for BA.4/BA.5,and 0.23 vs 0.12 for BA.4.6/BF.7) and even more so inBA.4/BA.5-convalescents for both the BA.4/BA.5 pseudovirus (GMT ratio0.48, p<0.01 versus BNT162b2³) and the BA.4.6/BF.7 pseudovirus (GMTratio 0.41, p<0.0001). Cross-neutralization of BA.4.6/BF.7 was alsosignificantly (p<0.0001) stronger in BA.4/BA.5 convalescents compared toquadruple-vaccinated individuals. While BQ.1.1 was cross-neutralizedless efficiently than BA.4/5 in all cohorts (GMT ratios 50.14),cross-neutralization in BA.4/BA.5 and BA.2 convalescents remainedsignificantly stronger compared to SARS-CoV-2 naïve triple or quadruplevaccinated and BA.1 breakthrough infected cohorts. Cross-neutralizationof BA.2.75, BA.2.75.2, and XBB pseudoviruses was broadly comparableacross cohorts. Together these data show that partial neutralization ofsome Omicron sublineages is retained especially in BA.4/BA.5convalescent individuals, and that neutralizating antibody responsesobserved in vaccinated and breakthrough infected individuals appeared tobe less effective against sublineases BA.2.75.2 and XBB. These data alsosuggest that, in some embodiments, an improved immune response (e.g.,increased neutralization titers) may be provided by a vaccine comprisingRNA encoding a SARS-CoV-2 S protein comprising one or more mutationscharacteristic of an XBB variant, a BQ.1.1 variant, or any lineagederived therefrom).

Example 23: T Cell Epitope and Neutralizing B Cell Epitope ConservednessAcross SARS-CoV-2 Variants of Concern

To estimate the rate of nonsynonymous mutation in T cell epitopes in theS glycoprotein, the Immune Epitope Database (https://www.iedb.org/) wasused to obtain epitopes confirmed for T cell reactivity in experimentalassays. The database was filtered using the following criteria:Organism: SARS-COV2; Antigen: Spike glycoprotein; Positive Assay; No Bcell assays; No MHC assays; MHC Restriction Type: Class I; Host: Homosapiens (human). The resulting table was filtered by removing epitopesthat were “deduced from a reactive overlapping peptide pool”, as well asepitopes longer than 14 amino acids in order to restrict the dataset toconfirmed minimal epitopes only. The experimental assays confirming thereactivity of these epitopes relied on multimer analysis, ELISpot orELISpot-like assays, T cell activation assays, etc. The epitopes werereported for at least 27 different HLA-I alleles, including HLA-A,HLA-B, and HLA-C alleles. Of the 251 unique epitope sequences obtainedin this approach, 244 were found in the Wuhan strain spike glycoprotein.Of these, 36 epitopes (14.8%) included a position reported to be mutatedby sequence analysis described herein.

In addition, conservedness of neutralizing B cell epitopes wascalculated by counting the number of antibody neutralising epitopespotentially impacted by mutations. It was calculated by first mapping719 binding epitopes observed in 332 experimentally resolved structuresof nAbs onto the S protein using available protein structures (asdescribed inhttps://www.biorxiv.org/content/10.1101/2021.12.24.474095v2). For eachstructure, an epitope per antibody was calculated as the set ofpositions that were in contact with an antibody, where two residues wereconsidered to be in contact if the smallest Euclidean distance betweentheir atoms was smaller than 4 Angstroms. Each nAb was evaluated andconsidered to be evaded by a variant if any position of its epitope wasmutated.

Given that humoral and cell-mediated immunity together determinesusceptibility to severe COVID-19 disease, the degree of conservationamong neutralizing B-cell and T-cell epitopes of the S glycoprotein inOmicron sublineages was assessed. The findings presented herein showthat over 80% of the BNT162b2 encoded S glycoprotein T-cell epitopeswere fully conserved in Omicron sublineages including BA.2.75.2, BQ.1.1,and XBB (FIG. 66 ). In striking contrast, B-cell epitopes were partiallyconserved in the earlier variants Alpha, Beta, and Delta (250%) but mostof these epitopes were altered in the Omicron lineage (520%conservation), particularly in BA.2.75.2 and XBB (510%). These findingssuggest that in the vaccinated population robust T-cell mediatedimmunity may be maintained against the Omicron variant, includingsublineages that evade neutralizing antibodies.

1. A composition or medical preparation comprising an RNA comprising anucleotide sequence encoding a SARS-CoV-2 Spike (S) protein comprising(a) one or more mutations characteristic of a SARS-CoV-2 Omicron variantand (b) an amino acid sequence that is at least 97% identical to SEQ IDNO: 49, or an immunogenic fragment thereof.
 2. The composition ormedical preparation of claim 1, wherein the SARS-CoV-2 S proteincomprises at least 10 of the following mutations: A67V, Δ69-70, T95I,G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F,K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y,Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H,N969K, and L981F, as compared to SEO ID NO:
 1. 3. The composition ormedical preparation of claim 2, wherein: the nucleotide sequenceencoding the SARS-CoV-2 S protein comprises a nucleotide sequence thatis at least 80% identical to SEQ ID NO: 50; and/or the RNA comprises anucleotide sequence that is at least 80% identical to SEQ ID NO:
 51. 4.The composition or medical preparation of claim 3, wherein theSARS-CoV-2 S protein comprises one or more proline mutations atpositions corresponding to one or more of residues 986 and 987 of SEQ IDNO:
 1. 5. The composition or medical preparation of claim 3, wherein theRNA comprises a modified nucleoside in place of uridine.
 6. Thecomposition or medical preparation of claim 5, wherein the RNA comprisesa modified nucleoside in place of each uridine, wherein the modifiednucleoside is N1-methyl-pseudouridine (m1ψ).
 7. The composition ormedical preparation of claim 3, wherein the RNA comprises one or more ofthe following: (a) a 5′-cap that is or comprises m₂ ^(7,3′-O)Gppp(m₁^(2′-O))ApG; (b) a poly(A) sequence comprising at least 100 Anucleotides; (c) a 5′-UTR that is or comprises a modified humanalpha-globin 5′-UTR; and (d) a 3′-UTR that is or comprises a firstsequence from the amino terminal enhancer of split (AES) messenger RNAand a second sequence from the mitochondrial encoded 12S ribosomal RNA.8. The composition or medical preparation of claim 3, wherein the RNA isformulated in lipid nanoparticles (LNP) comprising a cationicallyionizable lipid, a neutral lipid, a sterol and a polymer-lipidconjugate.
 9. The composition or medical preparation of claim 8, whereinthe RNA is present in an amount within a range of about 1 μg to about100 μg per dose.
 10. The composition or medical preparation of claim 9,wherein the RNA is present in an amount of about 1.5 μg, about 2.5 μg,about 3.0 μg, about 5.0 μg, about 10 μg, about 15 μg, about 30 μg, orabout 60 μg per dose.
 11. A composition or medical preparationcomprising: (a) a first RNA comprising a nucleotide sequence encoding aSARS-CoV-2 Spike (S) protein of a first strain or variant, or animmunogenic fragment thereof, and (b) a second RNA comprising anucleotide sequence encoding a SARS-CoV-2 Spike (S) protein of a secondvariant, or an immunogenic fragment thereof, wherein the first strain orvariant is different from the second variant, and wherein at least oneof the first RNA and the second RNA comprises a nucleotide sequenceencoding a SARS-CoV-2 S protein that comprises (i) one or more mutationscharacteristic of a SARS-CoV-2 Omicron variant and (ii) an amino acidsequence that is at least 97% identical to SEQ ID NO: 49, or animmunogenic fragment thereof.
 12. The composition or medical preparationof claim 11, wherein at least one of the first RNA and the second RNAcomprises a nucleotide sequence encoding a SARS-CoV-2 S proteincomprising at least 10 of the following mutations: A67V, Δ69-70, T95LG142D, Δ143-145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F,K417N, N440K, G446S, S477N, T478K, E484A, 0493R, G496S, 0498R N501Y,Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, 0954H,N969K, and L981F, as compared to SEO ID NO:
 1. 13. A composition ormedical preparation comprising a first RNA and a second RNA, wherein: a)the first RNA comprises a nucleotide sequence encoding a SARS-CoV-2 Sprotein of a Wuhan strain wherein (i) the SARS-CoV-2 S protein of aWuhan strain comprises an amino acid sequence that is at least 90%identical to SEQ ID NO: 7, and/or (ii) the first RNA comprises anucleotide sequence of that is at least 80% identical to SEQ ID NO: 9 or20, and b) the second RNA comprises a nucleotide sequence encoding aSARS-CoV-2 S protein comprising one or more mutations characteristic ofa BA.4/5 Omicron variant, wherein (i) the SARS-CoV-2 S proteincomprising one or more mutations characteristic of a BA.4/5 Omicronvariant comprises an amino acid sequence that is at least 90% identicalto SEQ ID NO: 69 and/or (ii) the second RNA comprises a nucleotidesequence that is at least 80% identical to SEQ ID NO: 70 or
 72. 14. Thecomposition or medical preparation of claim 13, wherein the SARS-CoV-2 Sprotein encoded by each of the first RNA and the second RNA comprisesone or more of the following features: (a) proline mutations at one ormore positions corresponding to residues 817, 892, 899, 942, 986, and987 of SEQ ID NO: 1; (b) a mutation that prevents furin cleavage,wherein the mutation is at a location corresponding to residues 682-685of SEQ ID NO: 1; and (c) an aspartate to glycine mutation at a positioncorresponding to residue 614 of SEQ ID NO:
 1. 15. The composition ormedical preparation of claim 13, wherein the first RNA and the secondRNA each comprise a modified nucleoside in place of uridine.
 16. Thecomposition or medical preparation of claim 15, wherein the first RNAand the second RNA each comprise a modified nucleoside in place of eachuridine, wherein the modified nucleoside is N1-methyl-pseudouridine(m1ψ).
 17. The composition or medical preparation of claim 13, whereinthe first RNA and the second RNA each comprise one or more of thefollowing features: (a) a 5′-cap that is or comprises m₂^(7,3′-)OGppp(m₁ ^(2′-O))ApG. (b) a poly(A) sequence that comprises 30adenine nucleotides followed by 70 adenine nucleotides, wherein the 30adenine nucleotides and 70 adenine nucleotides are separated by a linkersequence; (c) a 5′-UTR that is or comprises a modified humanalpha-globin 5′-UTR; and (d) a 3′-UTR that is or comprises a firstsequence from the amino terminal enhancer of split (AES) messenger RNAand a second sequence from the mitochondrial encoded 12S ribosomal RNA.18. The composition or medical preparation of claim 13, wherein thefirst RNA and the second RNA are each formulated as lipid nanoparticles(LNP), wherein the LNP comprise a cationically ionizable lipid, aneutral lipid, a sterol and a polymer-lipid conjugate.
 19. Thecomposition or medical preparation of claim 18, wherein the first RNAand the second RNA are formulated in separate lipid nanoparticles or inthe same lipid nanoparticles.
 20. The composition or medical preparationof claim 18, wherein the first RNA and the second RNA are present in acombined amount within a range of about 1 μg to about 100 μg per dose.21. The composition or medical preparation of claim 18, wherein: a) thefirst RNA and the second RNA are each present in an amount of about 1.5μg per dose in the composition; b) the first RNA and the second RNA areeach present in an amount of about 5 μg per dose in the composition; c)the first RNA and the second RNA are each present in an amount of about15 μg per dose in the composition; or d) the first RNA and the secondRNA are each present in an amount of about 30 μg per dose in thecomposition.
 22. The composition or medical preparation of claim 13,further comprising an RNA encoding one or more T cell epitopes ofSARS-CoV-2.
 23. The composition or medical preparation of claim 18,wherein the composition or medical preparation is formulated or is to beformulated for intramuscular administration.
 24. A method of elicitingan immune response against SARS-CoV-2 in a subject comprisingadministering the composition or medical preparation of claim
 18. 25.The method of claim 24, wherein the subject has previously beenadministered two or more doses of RNA encoding a SARS-CoV-2 S protein ofa Wuhan strain.
 26. The method of claim 24, wherein the first RNA andthe second RNA are formulated in the same lipid nanoparticles.
 27. Themethod of claim 24, further comprising administering an RNA comprising anucleotide sequence encoding one or more T cell epitopes of SARS-CoV-2.28. The method of claim 27, wherein the first RNA, the second RNA, andthe RNA comprising the nucleotide sequence encoding one or more T cellepitopes of SARS-CoV-2 are administered in a dose comprising: (a) 15 μgof the first RNA, 15 μg of the second RNA, and 5 μg of the RNAcomprising the nucleotide sequence encoding one or more T cell epitopesof SARS-CoV-2; (b) 15 μg of the first RNA, 15 μg of the second RNA, and10 μg of the RNA comprising the nucleotide sequence encoding one or moreT cell epitopes of SARS-CoV-2; or (c) 15 μg of the first RNA, 15 μg ofthe second RNA, and 15 μg of the RNA comprising the nucleotide sequenceencoding one or more T cell epitopes of SARS-CoV-2.
 29. The method ofclaim 24, further comprising administering one or more vaccines againsta respiratory infectious disease, wherein the respiratory infectiousdisease is RSV or influenza.